CN117377694A

CN117377694A - Protein comprising CD3 antigen binding domain and uses thereof

Info

Publication number: CN117377694A
Application number: CN202280037717.6A
Authority: CN
Inventors: A·兹沃拉克; R·布里廷汉姆; S·R·布罗德; R·加内桑; S·L·拉波特; 罗金泉; F·伊; C·M·凯恩; T·K·巴特
Original assignee: Janssen Biotech Inc
Current assignee: Janssen Biotech Inc
Priority date: 2021-03-24
Filing date: 2022-03-23
Publication date: 2024-01-09
Also published as: CA3214307A1; CL2023002804A1; CO2023012586A2; EP4314056A1; KR20230160353A; US12084501B2; AU2022242125A1; JP2024510777A; BR112023019458A2; TW202304987A; WO2022201053A1; US20230040715A1; AR125212A1; IL306132A; MX2023011266A

Abstract

The present disclosure provides antigen binding domains that bind cluster of differentiation 3 (CD 3), proteins comprising the antigen binding domains that bind CD3 epsilon, polynucleotides encoding them, vectors, host cells, methods of making and using them.

Description

Protein comprising CD3 antigen binding domain and uses thereof

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application Ser. No. 63/165,184 filed on 3/24 of 2021. The entire contents of the above-mentioned application are incorporated herein by reference in their entirety.

Electronically submitted reference sequence listing

The present application contains a sequence listing submitted electronically via EFS-Web as an ASCII formatted sequence listing, file name "JBI6516 WORTV1_SL.txt", creation date 2022, 3 months 17, size 1,282KB. This sequence listing submitted via EFS-Web is part of this specification and is incorporated by reference herein in its entirety.

Technical Field

The present disclosure provides antigen binding domains that bind cluster of differentiation 3 (CD 3) proteins, proteins comprising antigen binding domains that bind CD3, polynucleotides encoding them, vectors, host cells, methods of making and using them.

Background

Bispecific antibodies and antibody fragments have been explored as a means of recruiting cytolytic T cells to kill tumor cells. However, the clinical usefulness of many bispecific antibodies that recruit T cells is limited by challenges including adverse toxicity, potential immunogenicity, and manufacturing issues. Thus, there is a great need for improved bispecific antibodies that recruit cytolytic T cells to kill tumor cells including, for example, reduced toxicity and advantageous manufacturing characteristics.

The human CD 3T cell antigen receptor protein complex consists of six distinct chains: one CD3 gamma chain (SwissProt P09693), one CD3 delta chain (SwissProt P04234), two CD3 epsilon chains (SwissProt P07766) and one CD3 zeta chain homodimer (SwissProt P20963) (epsilon gamma: εδ: ζζ) associated with the T cell receptor alpha and beta chains. The complex plays an important role in coupling antigen recognition to several intracellular signal transduction pathways. The CD3 complex mediates signal transduction, leading to T cell activation and proliferation. CD3 is required for immune responses.

Redirecting cytotoxic T cells to kill tumor cells has become an important therapeutic mechanism for many tumor indications (Labrijn, A.F., janmaat, M.L., reichert, J.M., and Parren, P.), bispecific antibodies: a mechanistic review of the pipeline. Nat. Rev Drug discovery 18,585-608, doi:10.1038/s41573-019-0028-1 (2019)). T cell activation follows the double signal hypothesis, where a first signal is provided by engagement of a T Cell Receptor (TCR) complex with its cognate peptide MHC complex on an Antigen Presenting Cell (APC), and a second signal may be co-stimulatory or co-inhibitory (Chen, l. And Flies, d.b., molecular mechanisms of T cell co-stimulation and co-inhibition. Nat Rev Immunol 13,227-242, doi:10.1038/nri3405 (2013)). Tumors often do not present enough non-self antigen to induce a T cell-based immune response, whereas BsAbs (bsTCE) that engage T cells can overcome this challenge by inducing T cell activation without TCR-pMHC interactions. T cell receptor signaling occurs through ITAM motifs in the cytoplasmic region of the CD3 subunit of TCR (Chen, D.S. and Melman, I., oncology meets immunology: cancer-immunity cycle.immunity 39,1-10, doi: 10.1016/j.immunity.2013.07.012 (2013)). In particular, the CD3 epsilon subunit exists in two copies per TCR complex and represents an attractive antigen for T cell engagement. Indeed, many CD 3-targeted bsTCE epsilon show clinical anti-tumor efficacy in the event of mAb failure, and significant drug development efforts are underway against several tumor targets (Labrijn, a.f. et al, 2019). Three major challenges in clinical development of bsTCE are: 1) the possibility of rapid and severe toxicity associated with cytokine release via systemic or non-tumor T cell activation, 2) practical challenges of formulation and administration of bsTCE with high potency and strong therapeutic index, and 3) the possibility of reactivation-induced T cell death, wherein tumor-infiltrating T cells (TILS) undergo apoptosis in response to excessive activation of bsTCE (Wu, z.wu and Cheung, n.v., T cell engaging bispecific antibody (T-BsAb): from technology to therapeutics. Pharmacol ter 182,161-175, doi:10.1016/j.pharmthera.2017.08.005 (2018)).

Taken together, these observations suggest a need in the art for novel CD 3-specific binding proteins that are more advantageous and useful in the treatment of cancer.

Disclosure of Invention

For example, the present disclosure meets this need by providing novel CD3 epsilon specific binding proteins with high affinity for tumor antigens and weak affinity for T cells. The proteins produced by the present disclosure that comprise an antigen binding domain that binds CD3 epsilon have high thermostability, reduced risk of deamidation, and are humanized to reduce immunogenicity.

In certain embodiments, the invention provides an isolated protein comprising an antigen binding domain that binds to cluster 3 epsilon (CD 3 epsilon), wherein the antigen binding domain that binds to CD3 epsilon comprises: an isolated protein comprising an antigen binding domain that binds to cluster 3 epsilon (CD 3 epsilon), wherein the antigen binding domain that binds to CD3 epsilon comprises:

heavy chain complementarity determining regions (HCDR) 1, HCDR2 and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO. 55, and light chain complementarity determining regions (LCDR) 1, LCDR2 and LCDR3 of the light chain variable region (VL) of SEQ ID NO. 59;

HCDR1, HCDR2 and HCDR3 of VH of SEQ ID No. 55, and LCDR1, LCDR2 and LCDR3 of VL of SEQ ID No. 58;

HCDR1, HCDR2 and HCDR3 of VH of SEQ ID No. 54, and LCDR1, LCDR2 and LCDR3 of VL of SEQ ID No. 56; or alternatively

HCDR1, HCDR2 and HCDR3 of VH of SEQ ID No. 48, and LCDR1, LCDR2 and LCDR3 of VL of SEQ ID No. 58;

wherein the amino acid in position N106 of SEQ ID NO. 55, 54 or 48 is optionally substituted with an amino acid selected from the group consisting of A, G, S, F, E, T, R, V, I, Y, L, P, Q and K, wherein the residue numbering starts from the N-terminus of SEQ ID NO. 55, 54 or 48.

In certain embodiments, the disclosure also provides an isolated protein comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as SEQ ID NOs 70, 71, 86, 79, 80, and 81, respectively.

In other embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are each

70, 71, 72, 79, 80 and 81;

70, 71, 87, 79, 80 and 81; or alternatively

SEQ ID NOS 70, 71, 90, 79, 80 and 81.

In other embodiments, the antigen binding domain that binds CD3 epsilon is an scFv, (scFv) 2, fv, fab, F (ab') 2, fd, dAb, or VHH.

In other embodiments, the antigen binding domain that binds CD3 epsilon is a Fab.

In other embodiments, the antigen binding domain that binds CD3 epsilon is an scFv.

In other embodiments, the scFv comprises, from N-terminus to C-terminus, a VH, a first linker (L1) and a VL (VH-L1-VL), or comprises a VL, L1 and a VH (VL-L1-VH).

In other embodiments, the L1 comprises:

about 5 to 50 amino acids;

about 5 to 40 amino acids;

about 10 to 30 amino acids; or alternatively

About 10 to 20 amino acids.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NOS 3 to 36.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 3.

In other embodiments, the antigen binding domain that binds CD3 epsilon comprises the VH of SEQ ID NO:55, 54 or 48, and the VL of SEQ ID NO:59, 58 or 56.

In other embodiments, the antigen binding domain that binds CD3 epsilon comprises:

VH of SEQ ID NO. 55 and VL of SEQ ID NO. 59;

VH of SEQ ID NO. 55 and VL of SEQ ID NO. 58;

VH of SEQ ID NO. 54 and VL of SEQ ID NO. 56;

VH of SEQ ID NO. 48 and VL of SEQ ID NO. 58;

the VH of SEQ ID NO. 88 and the VL of SEQ ID NO. 58; or alternatively

The VH of SEQ ID NO. 242 and the VL of SEQ ID NO. 58.

In other embodiments, the antigen binding domain that binds CD3 epsilon comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 96 to 126.

In other embodiments, the isolated protein is a multispecific protein.

In other embodiments, the multispecific protein is a bispecific protein.

In other embodiments, the multispecific protein is a trispecific protein.

In other embodiments, the isolated protein further comprises an immunoglobulin (Ig) constant region or a fragment of the Ig constant region.

In other embodiments, a fragment of the Ig constant region comprises an Fc region.

In other embodiments, a fragment of the Ig constant region comprises a CH2 domain.

In other embodiments, a fragment of the Ig constant region comprises a CH3 domain.

In other embodiments, the fragment of the Ig constant region comprises a CH2 domain and a CH3 domain.

In other embodiments, a fragment of the Ig constant region comprises at least a portion of a hinge, a CH2 domain, and a CH3 domain.

In other embodiments, the fragment of the Ig constant region comprises a hinge, a CH2 domain, and a CH3 domain.

In other embodiments, the antigen binding domain that binds CD3 epsilon is conjugated to the N-terminus of an Ig constant region or fragment of an Ig constant region.

In other embodiments, the antigen binding domain that binds CD3 epsilon is conjugated to the C-terminus of an Ig constant region or fragment of an Ig constant region.

In other embodiments, the antigen binding domain that binds CD3 epsilon is conjugated to an Ig constant region or fragment of an Ig constant region via a second linker (L2).

In other embodiments, the L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 3 to 36.

In other embodiments, the multispecific protein comprises an antigen-binding domain that binds an antigen other than CD3 epsilon.

In other embodiments, the cellular antigen is a tumor-associated antigen.

In other embodiments, the Ig constant region or fragment of an Ig constant region is an IgG1, igG2, igG3, or IgG4 isotype.

In other embodiments, the Ig constant region or fragment of the Ig constant region comprises at least one mutation that results in reduced binding of the protein to an fcγ receptor (fcγr).

In other embodiments, the at least one mutation that results in reduced binding of the protein to fcγr is selected from the group consisting of: F234A/L235A, L A/L235A, L A/L235A/D265S, V234A/G237A/P238S/H268A/V309L/A330S/P331S, F A/L235A, S P/F234A/L235A, N297A, V A/G237A, K T/E233P/L234V/L235A/G236-deletion/A327G/P331A/D365E/L358M, H268Q/V309L/A330S/P331S, S E/L328F, L F/L235E/D265A, L A/L235A/G237A/P238S/H268A/A330S/P331S, S P/F234A/L235A/G237A/P238S and S228P/F234A/L235A/G236-deletion/G237A/P238S, wherein residue numbering is performed according to the EU index.

In other embodiments, the fcγr is fcγri, fcγriia, fcγriib, or fcγriii, or any combination thereof.

In other embodiments, the protein comprises at least one mutation in the CH3 domain of the Ig constant region.

In other embodiments, the at least one mutation in the CH3 domain of the Ig constant region is selected from the group consisting of: T350V, L351Y, F A, Y407V, T366Y, T366W, T366L, T L, F405W, T394W, K392L, T394S, T394W, Y407T, Y A, T S/L368A/Y407V, L Y/F405A/Y407V, T I/K392M/T394W, F A/Y407V, T366L/K392M/T394W, T L/K392L/T394W, L57351Y/Y407A, L Y/Y407V, T A/K409F, T366V/K409F, T366A/K409F, T V/L351Y/F405A/Y407V and T350V/T366L/K392L/T394W, wherein residue numbering is according to EU index.

The present disclosure also provides a pharmaceutical composition comprising the isolated protein and a pharmaceutically acceptable carrier.

The present disclosure also provides a polynucleotide encoding the isolated protein.

The present disclosure also provides a vector comprising the polynucleotide.

The present disclosure also provides a host cell comprising the vector.

The present disclosure also provides a method of producing the isolated protein comprising culturing the host cell under conditions such that the protein is expressed, and recovering the protein produced by the host cell.

The present disclosure also provides a method of treating cancer in a subject comprising administering to a subject in need thereof a therapeutically effective amount of an isolated protein to treat cancer.

The present disclosure also provides an anti-idiotype antibody that binds to the isolated protein.

The present disclosure also provides an isolated protein of any one of claims 1 to 35 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 127 to 157.

The present disclosure also provides an isolated protein comprising an antibody heavy chain of SEQ ID NO. 224 and an antibody light chain of SEQ ID NO. 226.

Drawings

The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the antibodies and methods of the present disclosure, there are shown in the drawings exemplary embodiments of the antibodies and methods; however, the antibodies and methods are not limited to the specific embodiments disclosed. In the drawings:

FIG. 1 shows the binding of murine Cris-7 (CD 3B1127 and CD 31128) and human germline graft Cris-7 variant sequences in the form of scFv, as determined by ELISA.

FIGS. 2A and 2B show the percentage of E.coli expressed scFv clones retaining at least 75% binding as determined by ELISA after heat shock at 60℃for either humanized VH paired with murine VL (2A) or humanized VL paired with murine VH (2B); the numbers on the X-axis represent residue positions.

FIG. 3 shows the results of comparison of binding capacities of humanized CD 3-specific scFv containing human-mouse back mutation based on ELISA.

FIG. 4 shows binding of scFv-form CD3B2030 variants to recombinant CD3 (TRCW 5) as determined by ELISA; "NtoX" indicates an amino acid substitution at position 106 of VH (SEQ ID NO: 55), wherein "X" is the amino acid indicated in the accompanying drawings.

FIG. 5 shows the hydrogen-deuterium exchange rates determined using hydrogen-deuterium exchange mass spectrometry (HDX-MS) of: the Cris7 complex (bivalent or monovalent) that binds to human CD3 epsilon, or the OKT3 complex (CD 3 epsilon: OKT 3) that binds to human CD3 epsilon (fragments of CD3 epsilon (SEQ ID NO: 1) are shown). Fragments whose deuteration level was reduced by more than 30% compared to CD3 epsilon alone in the presence of antibody are underlined. FIG. 5 discloses SEQ ID NOS 1508, 1509 and 1509, respectively, in the order of appearance.

FIG. 6 depicts a depiction of exemplary CD79b.times.CD20.times.CD3 trispecific antibodies.

Fig. 7A to 7D. Binding affinity of selected CD79bxCD3 bsAb in the following cell lines: HLB-1 cell line (FIG. 7A); the OCI-LY10 cell line (FIG. 7B); a carnval cell line (fig. 7C); WILL-2 cell line (FIG. 7D). The circle corresponds to 79C3B646 bsAb; triangles correspond to 79C3B651 bsAb; the diamonds correspond to 79C3B601 bsAb.

Fig. 8A to 8D. Binding affinity of selected CD79bxCD20xCD3 trispecific antibodies in the following cell lines: HLB-1 cell line (FIG. 8A); the OCI-LY10 cell line (FIG. 8B); a carnval cell line (fig. 8C); WILL-2 cell line (FIG. 8D). Filled circles correspond to 79C3B646 bsAb controls; solid triangles correspond to 79C3B651 bsAb control; filled diamonds correspond to 79C3B601 bsAb controls. The open triangles correspond to trispecific antibody C923B38; open diamonds correspond to trispecific antibody C923B74; asterisks correspond to trispecific antibody C923B9; x corresponds to the control trispecific antibody C923B98.

Fig. 9A to 9I. Binding kinetics of selected CD79bxCD3 bsAb on DLBCL cell lines. Binding kinetics at 300nm for three selected bsAb in HBL-1 cells (fig. 9A). Binding kinetics of three selected bsAb at 60nm in HBL-1 cells (fig. 9B). Binding kinetics at 12nm for three selected bsAb in HBL-1 cells (fig. 9C). Binding kinetics of three selected bsabs at 300nm in carnval cells (fig. 9D). Binding kinetics of three selected bsabs at 60nm in carnval cells (fig. 9E). Binding kinetics of three selected bsabs at 12nm in carnval cells (fig. 9F). Binding kinetics at 300nm for three selected bsAb in OCI-LY10 cells (fig. 9G). Binding kinetics at 60nm for three selected bsAb in OCI-LY10 cells (fig. 9H). Binding kinetics at 12nm for three selected bsAb in OCI-LY10 cells (fig. 9I). The inverted triangle corresponds to 79C3B646 bsAb; diamonds correspond to 79C3B651 bsAb; the square corresponds to 79C3B601 bsAb.

Fig. 10A to 10I. Binding kinetics of selected CD79bxCD20xCD3 trispecific antibodies on DLBCL cell lines. Binding kinetics of selected antibodies in HBL-1 cells at 300nm (fig. 10A). Binding kinetics of selected antibodies in HBL-1 cells at 60nm (FIG. 10B). Binding kinetics of selected antibodies in HBL-1 cells at 12nm (fig. 10C). Binding kinetics of selected antibodies in carnval cells at 300nm (fig. 10D). Binding kinetics of selected antibodies in carnval cells at 60nm (fig. 10E). Binding kinetics of selected antibodies in carnval cells at 12nm (fig. 10F). Binding kinetics of selected antibodies in OCI-LY10 cells at 300nm (fig. 10G). Binding kinetics of selected antibodies in OCI-LY10 cells at 60nm (FIG. 10H). Binding kinetics of selected antibodies in OCI-LY10 cells at 12nm (fig. 10I). The inverted triangle corresponds to the 79C3B646 bsAb control; diamonds correspond to 79C3B651 bsAb control; squares correspond to 79C3B601 bsAb controls. Triangles correspond to trispecific antibody C923B38; the circle corresponds to trispecific antibody C923B74; the squares correspond to trispecific antibody C923B99; asterisks correspond to the blank trispecific antibody C923B98.

Fig. 11A to 11D. Primary pan T cell binding of CD79bxCD20xCD3 trispecific antibody and CD79bxCD3 bispecific antibody. Binding kinetics of selected antibodies in the pan T cell donor line D221837 (fig. 11A). Binding kinetics of selected antibodies in the pan T cell donor line D329312 (fig. 11B). Binding kinetics of selected antibodies in the pan T cell donor line D329335 (fig. 11C). Binding kinetics of selected antibodies in the pan T cell donor line D160115 (fig. 11D). The circle corresponds to 79C3B651 bsAb; squares correspond to 79C3B646bsAb; triangles correspond to trispecific antibody C923B38; the inverted triangle corresponds to the trispecific antibody C923B99; diamonds correspond to trispecific antibody C923B74.

Fig. 12A-12 b.t cell cytotoxicity of cd79bxCD20xcd3 trispecific antibody and CD79bxCD3 bispecific antibody. Cytotoxicity of selected antibodies in HEL T cell lines (fig. 12A). Cytotoxicity of selected antibodies in the K562T cell line (fig. 12B). The shaded circle corresponds to trispecific antibody C923B74; blank circles correspond to trispecific antibody C923B99; triangles correspond to trispecific antibody C923B38; the inverted triangle corresponds to 79C3B646bsAb; diamonds correspond to 79C3B651 bsAb; black squares correspond to 79C3B601 bsAb; white squares correspond to C923B98 bsAb.

FIGS. 13A through 13℃ CD79bxCD20xCD3 trispecific construct mediated B cell cytotoxicity and T cell activation. Shows cytotoxicity of the lead antibody in B cells (FIG. 13A), at CD4 ⁺ Cytotoxicity in T cells (fig. 13B), and in CD8 ⁺ Cytotoxicity in T cells.

Figure 14 shows HDX-MS epitope mapping of PSMA for PS3B1352 (top) and PS3B1353 (bottom). G is a glycosylation site. Black boxes are epitopes and grey is potential epitope. White boxes indicate no/little change in deuteration level in the presence of antibody. Residues without boxes indicate that HDX behavior is not monitored, as no peptide covers the residues or the residues are the first two residues of the peptide. The epitopes of PS3B1352 and PS3B1353 are identical. These epitopes are residues 597-598 (CR) because fragments 597-598 are significantly protected after binding (average difference in deuteration levels is more than 10%). These epitopes include residues 599 (D), 602-603 (VV) and 605 (R) because they are weakly protected (average difference in deuteration levels is 5% to 10%) when bound and may show greater protection if the time point monitored is longer. These epitopes may be larger than the four fragments, since fragments surrounding the four fragments, such as 593-594 (LP), 595 (F), 596 (D), 600 (Y), 601 (a), 604 (L), 606-607 (KY), 607-609 (YAD) and 610-612 (KIY), do not exchange at all within the time window employed, and may be protected if the monitored time point is longer. FIG. 14 discloses SEQ ID NO 1510.

Figure 15 shows HDX-MS identified PSMA epitopes overlaid on X-ray crystal structure. Blue: an epitope; sky blue: possible epitopes; blue-green: potential epitopes.

Fig. 16 shows a pan T cell binding assay. Human pan T cells were treated with various concentrations of PSMA/CD3 bispecific antibody and incubated at 37 ℃ for 30 min, followed by CD3 cell surface expression analysis by flow cytometry.

Figure 17 shows a non-linear regression fit of the four parameter function of PSMA ligand binding for C4-2B human prostate tumor cells.

Fig. 18 shows a target cell binding assay. C4-2B human prostate tumor cells were treated with different concentrations of PSMA/CD3 bispecific antibody and incubated at 37℃for 30 min, followed by analysis of PSMA cell surface expression by flow cytometry.

Fig. 19 illustrates internalization of PSMA. Conjugation of human C4-2B prostate tumor cells toThe PSMA/CD3 bispecific antibody of the Human Fab-fluor-pH red antibody labeling dye was incubated together for 24 hours.

Fig. 20A to 20H show the following stepsBispecific anti-PSMA/anti-T cell redirecting antibodies evaluated in cytotoxicity assays of (a). The isolated pan T cells were incubated with psma+c4-2B cells for 120 hours in the presence of bispecific PSMA/T cell redirecting antibodies. Data for (a) PS3B1352, (B) PS3B1356, (C) PS3B1353, (D) PS3B1357, (E) PS3B1354, (F) PS3B937, (G) PS3B1355, and (H) PS3B1358 are shown.

Figure 21 shows a T cell redirecting killing assay. PBMC from normal human and useNucLight red nuclear dye transduced C4-2B human prostate tumor cells were pooled and treated with PSMA/CD3 bispecific antibody for 5 days.

Figure 22 shows cytokine induction by bispecific anti-PSMA/anti-T cell redirecting antibodies. Isolated pan T cells were co-incubated with psma+c4-2B cells in the presence of bispecific anti-PSMA/anti-T cell redirecting antibodies for a designated time point. The IFN-y concentration in supernatants collected at the indicated time points was measured.

Detailed Description

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if fully set forth herein.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

When a list is provided, it is to be understood that each individual element in the list and each combination of the list is a separate embodiment unless indicated otherwise. For example, a list of embodiments presented as "A, B or C" will be understood to include embodiments "a", "B", "C", "a or B", "a or C", "B or C" or "A, B or C".

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and so forth.

The transitional terms "comprising," "consisting essentially of … …," and "consisting of … …" are intended to imply their accepted meanings in the patent literature; that is, (i) "comprises" is synonymous with "comprising," "contains," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; (ii) "consisting of … …" excludes any element, step or component not specified in the claims; and (iii) consist essentially of … …, limiting the scope of the claims to the materials or steps specified, as well as materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. Embodiments described in the phrase "comprising" (or equivalents thereof) are also provided, as are those embodiments described independently in terms of "consisting of … …" and "consisting essentially of … ….

"about" means within acceptable error limits of the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. In the context of a particular assay, result, or embodiment, unless otherwise explicitly stated in the examples or elsewhere in the specification, "about" means within one standard deviation or up to a range of 5% (whichever is greater) according to convention in the art.

"activating" or "stimulation" or "activated" or "stimulated" refers to inducing a change in a biological state of a cell, resulting in expression of an activation marker, cytokine production, proliferation, or mediating cytotoxicity of a target cell. Cells can be activated by a primary stimulus signal. The co-stimulatory signal may amplify the amplitude of the primary signal and suppress cell death following initial stimulation, thereby producing a more durable activation state and thus a higher cytotoxic capacity. "costimulatory signal" refers to a signal that, in combination with a primary signal (such as a TCR/CD3 linkage), results in up-or down-regulation of T cell and/or NK cell proliferation and/or key molecules.

"surrogate scaffold" refers to a single-chain protein framework comprising a structured core associated with a high conformational tolerance variable domain. The variable domains are tolerant of the changes to be introduced without compromising scaffold integrity, and thus can be engineered and selected to bind to specific antigens.

"cytotoxicity of antibody-dependent cells", "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a mechanism that induces cell death that depends on the interaction of antibody-coated target cells with effector cells having lytic activity, such as natural killer cells (NK), monocytes, macrophages and neutrophils, via fcγr expressed on the effector cells.

"antibody-dependent cellular phagocytosis" or ADCP refers to a mechanism by which antibody-coated target cells are eliminated by internalization of phagocytes, such as macrophages or dendritic cells.

An "antigen" refers to any molecule (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portion thereof, or combination thereof) capable of binding by an antigen binding domain or a T cell receptor capable of mediating an immune response. Exemplary immune responses include antibody production and activation of immune cells such as T cells, B cells or NK cells. The antigen may be gene expressed, synthesized or purified from biological samples such as tissue samples, tumor samples, cells or fluids with other biological components, organisms, subunits of proteins/antigens, killed or inactivated whole cells or lysates.

"antigen binding fragment" or "antigen binding domain"Refers to the antigen-binding portion of a protein. The antigen binding domain may be a synthetic, enzymatically obtainable or genetically engineered polypeptide and includes antigen-binding portions of immunoglobulins such as VH, VL, VH and VL, fab, fab ', F (ab') ₂ Fd and Fv fragments; a domain antibody (dAb) consisting of one VH domain or one VL domain; a shark variable IgNAR domain; humping the VH domain; a VHH domain; a minimal recognition unit consisting of amino acid residues of CDRs such as FR3-CDR3-FR4 portions, HCDR1, HCDR2 and/or HCDR3, LCDR1, LCDR2 and/or LCDR3 of the mimetic antibody; an alternative scaffold that binds antigen; and multispecific proteins comprising antigen-binding fragments. Antigen binding fragments, such as VH and VL, can be joined together via synthetic linkers to form various types of single antibody designs, wherein in those cases where the VH and VL domains are expressed from separate single chains, the VH/VL domains can be paired intramolecularly or intermolecularly to form monovalent antigen binding sites, such as single chain Fv (scFv) or diabodies. Antigen binding fragments may also be conjugated to other antibodies, proteins, antigen binding fragments, or alternative scaffolds, which may be monospecific or multispecific to engineer bispecific and multispecific proteins.

"antibody" broadly refers to and includes immunoglobulin molecules, particularly including monoclonal antibodies (including murine monoclonal antibodies, human monoclonal antibodies, humanized monoclonal antibodies, and chimeric monoclonal antibodies), antigen binding fragments, multispecific antibodies (such as bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and the like), dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies, and any other modified configuration of immunoglobulin molecules comprising an antigen binding site having the desired specificity. "full length antibodies" comprise two Heavy Chains (HC) and two Light Chains (LC) interconnected by disulfide bonds and multimers thereof (e.g., igM). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (consisting of domains CH1, hinge, CH2 and CH 3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions may be further subdivided into regions of hypervariability termed Complementarity Determining Regions (CDRs) interspersed with Framework Regions (FR). Each VH and VL is made up of three CDRs and four FR segments, and arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Immunoglobulins can be assigned to five major classes, igA, igD, igE, igG and IgM, based on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified into isotypes IgA1, igA2, igG1, igG2, igG3 and IgG4. Based on the amino acid sequence of its constant domain, the antibody light chain of any spinal species can be assigned to one of two completely different types, namely kappa and lambda.

"bispecific" refers to molecules (such as proteins or antibodies) that specifically bind to two different antigens or two different epitopes within the same antigen. Bispecific molecules may be cross-reactive to other related antigens, for example, to the same antigen from other species (homologous), such as humans or monkeys, e.g., cynomolgus macaque (Macaca cynomolgus) (cynomolgus) or chimpanzees (Pan troglymes), or may bind to an epitope shared between two or more different antigens.

"bispecific anti-PSMA/anti-CD 3 antibody", "PSMA/CD3 antibody", "PSMAxCD3 antibody", "anti-PSMA/anti-CD 3 protein" and the like refer to antibodies that bind PSMA and CD3 and comprise at least one binding domain that specifically binds PSMA and at least one binding domain that specifically binds CD 3. The domain that specifically binds PSMA and CD3 is typically V _H /V _L For each pair. Bispecific anti-PSMA x CD3 antibodies can be monovalent in terms of their binding to PSMA or CD 3.

"bispecific anti-CD 79 b/anti-CD 3 antibody", "anti-CD 79b x CD3", "CD79b/CD3 antibody", "CD79bx CD3 antibody", "anti-CD 79 b/anti-CD 3 protein" and the like refer to antibodies that bind CD79b and CD3 and comprise at least one binding domain that specifically binds CD79b and at least one binding domain that specifically binds CD 3. The domain that specifically binds CD79b and CD3 is typically V _H /V _L For each pair. Bispecific anti-CD 79b x CD3 antibodies may be monovalent in terms of their binding to CD79b or CD 3.

"cancer" refers to a wide variety of diseases characterized by uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth results in the formation of malignant tumors that invade adjacent tissues, and may also metastasize to distal parts of the body through the lymphatic system or blood flow. "cancer" or "cancer tissue" may include tumors.

"cluster 3 ε" or "CD3 ε" refers to a known protein also known as "T cell surface glycoprotein CD3 ε chain", or "T3E". CD3 epsilon forms a T cell receptor-CD 3 complex with CD 3-gamma, CD 3-delta, and CD 3-zeta, and T cell receptor alpha/beta and gamma/delta heterodimers. The complex plays an important role in coupling antigen recognition to several intracellular signal transduction pathways. The CD3 complex mediates signal transduction, leading to T cell activation and proliferation. CD3 is required for immune responses. The amino acid sequence of full length CD3 epsilon is shown in SEQ ID NO. 1. The amino acid sequence of the extracellular domain (ECD) of CD3 epsilon is shown in SEQ ID NO. 2. Throughout the specification, "CD3 epsilon specific" or "specifically binds CD3 epsilon" or "anti-CD 3 antibody" refers to an antibody that specifically binds to the CD3 epsilon polypeptide (SEQ ID NO: 1), including an antibody that specifically binds to the CD3 epsilon extracellular domain (ECD) (SEQ ID NO: 2).

SEQ ID NO. 1 (human CD3 epsilon)

MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI

SEQ ID NO. 2 (human CD3 epsilon extracellular domain)

DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD

"cluster of differentiation CD79B protein" or "CD79B" refers to B cell antigen receptor (BCR) signaling component Igβ. Amino acid sequences for the various isoforms can be retrieved from GenBank accession nos. AAH32651.1, EAW94232.1, AAH02975.2, np_000617.1 and np_ 001035022.1. The amino acid sequence of the full length CD79b sequence is shown below (SEQ ID NO: 241). The sequence includes an extracellular domain (residues 29-159) and a cytoplasmic domain (residues 181-229).

MARLALSPVPSHWMVALLLLLSAEPVPAARSEDRYRNPKGSACSRIWQSPRFIARKRGFTVKMHCYMNSASGNVSWLWKQEMDENPQQLKLEKGRMEESQNESLATLTIQGIRFEDNGIYFCQQKCNNTSEVYQGCGTELRVMGFSTLAQLKQRNTLKDGIIMIQTLLIILFIIVPIFLLLDKDDSKAGMEEDHTYEGLDIDQTATYEDIVTLRTGEVKWSVGEHPGQE(SEQ ID NO:241)。

"complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death in which the Fc effector domain of a target binding protein binds to and activates complement component C1q, which in turn activates the complement cascade, resulting in target cell death. Activation of complement can also result in deposition of complement components on the target cell surface that promote CDC by binding to complement receptors (e.g., CR 3) on leukocytes.

"complementarity determining regions" (CDRs) are regions of an antibody that bind antigen. Three CDRs (HCDR 1, HCDR2, HCDR 3) are present in VH and three CDRs (LCDR 1, LCDR2, LCDR 3) are present in VL. The CDRs may be defined using various depictions such as Kabat (Wu et al, (1970) J Exp Med 132:211-50; kabat et al, sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, md., 1991), chothia (Chothia et al, (1987) J Mol Biol 196:901-17), IMGT (Lefranc et al, (2003) Dev Comp Immunol 27:55-77) and AbM (Martin and Thorton, J Bmol Biol 263:800-15,1996). The correspondence between various delineation and variable region numbering is described (see, e.g., lefranc et al, (2003) Dev Comp Immunol 27:55-77; honyger and Pluckaphun, J Mol Biol (2001) 309:657-70; international Immunogenetics (IMGT) database; network resources (e.g., retrievable from the Internet < URL: http:// www.imgt.org >)) available programs (such as abYsis of UCL Business PLC) may be used to delineate CDRs. As used herein, the terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2", and "LCDR3" include CDRs defined by any of the above methods (Kabat, chothia, IMGT or AbM), unless the specification clearly indicates otherwise.

"decrease", "abate", "decrease" or "attenuation" refers generally to the ability of a test molecule to mediate a reduced response (i.e., downstream effect) when compared to a response mediated by a control or vehicle. Exemplary responses are T cell expansion, T cell activation, or T cell mediated tumor cell killing or binding of proteins to their antigens or receptors, enhanced binding to fcγ or enhanced Fc effector functions such as enhanced ADCC, CDC and/or ADCP. The decrease may be a statistically significant difference in the measured response between the test molecule and the control (or vehicle), or a decrease in the measured response, such as a decrease of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30-fold or more, such as 500, 600, 700, 800, 900, or 1000-fold or more (including all integer and decimal points between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.).

"differentiation" refers to a method of reducing the potency or proliferation of a cell or bringing a cell into a more developmentally restricted state.

"coding" refers to the inherent properties of a particular nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) that serves as a template for the synthesis of other polymers and macromolecules in biological processes, which have defined nucleotide sequences (e.g., rRNA, tRNA, and mRNA) or defined amino acid sequences and biological properties resulting therefrom. Thus, if transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, the gene, cDNA or RNA encodes the protein. Both the coding strand (which has the same nucleotide sequence as the mRNA) and the non-coding strand (which serves as a template for transcription of the gene or cDNA) may be referred to as encoding the protein, or other product of the gene or cDNA.

"enhancing," "promoting," "increasing," "amplifying," or "ameliorating" generally refers to the ability of a test molecule to mediate a stronger response (i.e., downstream effect) when compared to a response mediated by a control or vehicle. Exemplary responses are T cell expansion, T cell activation, or T cell mediated tumor cell killing or binding of proteins to their antigens or receptors, enhanced binding to fcγ or enhanced Fc effector functions such as enhanced ADCC, CDC and/or ADCP. The enhancement may be a statistically significant difference in the measured response between the test molecule and the control (or vehicle), or an increase in the measured response, such as an increase of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30-fold or more, such as 500, 600, 700, 800, 900, or 1000-fold or more (including all integers and decimal points between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.).

An "epitope" refers to a portion of an antigen to which an antibody or antigen-binding portion thereof specifically binds. Epitopes are generally composed of chemically active (such as polar, nonpolar or hydrophobic) surface groups of moieties such as amino acids or polysaccharide side chains, and may have specific three-dimensional structural features as well as specific charge features. Epitopes can be composed of contiguous and/or non-contiguous amino acids that form conformational space units. For discontinuous epitopes, the amino acids from different parts of the linear sequence of the antigen are close in three dimensions due to folding of the protein molecule. Antibodies "epitopes" depend on the method used to identify the epitope.

"expansion" refers to the result of cell division and cell death.

"expression" refers to well known transcription and translation occurring in cells or in vitro. The expression product (e.g., protein) is expressed by the cell or expressed in vitro, and may be an intracellular protein, an extracellular protein, or a transmembrane protein.

An "expression vector" refers to a vector that can be used in a biological system or reconstituted biological system to direct translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

"dAb" or "dAb fragment" refers to an antibody fragment consisting of a VH domain (Ward et al Nature 341:544 546 (1989)).

"Fab" or "Fab fragment" refers to an antibody fragment consisting of the VH, CH1, VL and CL domains.

“F(ab') ₂ "OR" F (ab') ₂ Fragment "refers to an antibody fragment containing two Fab fragments linked by a disulfide bridge in the hinge region.

"Fd" or "Fd fragment" refers to an antibody fragment consisting of a VH and CH1 domain.

"Fv" or "Fv fragment" refers to an antibody fragment consisting of a VH domain and a VL domain from a single arm of an antibody.

"full length antibodies" comprise two Heavy Chains (HC) and two Light Chains (LC) interconnected by disulfide bonds and multimers thereof (e.g., igM). Each heavy chain is composed of a heavy chain variable domain (VH) and a heavy chain constant domain composed of subdomains CH1, hinge, CH2 and CH 3. Each light chain is composed of a light chain variable domain (VL) and a light chain constant domain (CL). VH and VL can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with Framework Regions (FR). Each VH and VL is made up of three CDRs and four FR segments, and arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

"genetic modification" refers to the introduction of a "foreign" (i.e., foreign or extracellular) gene, DNA or RNA sequence into a host cell such that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. The introduced gene or sequence may also be referred to as a "cloned" or "foreign" gene or sequence, and may include regulatory or control sequences operably linked to a polynucleotide encoding a chimeric antigen receptor, such as initiation, termination, promoters, signals, secretion, or other sequences used by the genetic machinery of the cell. The gene or sequence may include a nonfunctional sequence or a non-functional sequence. Host cells that receive and express the introduced DNA or RNA have been "genetically engineered". The DNA or RNA introduced into the host cell may be from any source, including cells of the same genus or species as the host cell, or from a different genus or species.

"heterologous" refers to two or more polynucleotides or two or more polypeptides that are not in the same relationship to each other in nature.

"heterologous polynucleotide" refers to a non-naturally occurring polynucleotide encoding two or more neoantigens as described herein.

By "heterologous polypeptide" is meant a non-naturally occurring polypeptide comprising two or more neoantigen polypeptides as described herein.

"host cell" refers to any cell containing a heterologous nucleic acid. Exemplary heterologous nucleic acids are vectors (e.g., expression vectors).

"human antibody" refers to an antibody that is optimized to have a minimal immune response when administered to a human subject. The variable regions of human antibodies are derived from human immunoglobulin sequences. If the human antibody comprises a constant region or a portion of a constant region, the constant region is also derived from a human immunoglobulin sequence. A human antibody comprises a heavy chain variable region and a light chain variable region "derived from" sequences of human origin if the variable region is obtained from a system using human germline immunoglobulins or rearranged immunoglobulin genes. Such exemplary systems are libraries of human immunoglobulin genes displayed on phage, as well as transgenic non-human animals, such as mice or rats carrying human immunoglobulin loci. Because of the differences between the systems used to obtain human antibodies and human immunoglobulin loci, the introduction of somatic mutations or the intentional substitution will be introduced into the framework or CDRs or both, and thus "human antibodies" typically comprise amino acid differences compared to immunoglobulins expressed in humans. Typically, the amino acid sequence of a "human antibody" has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence encoded by a human germline immunoglobulin gene or a rearranged immunoglobulin gene. In some cases, a "human antibody" may comprise a consensus framework sequence derived from human framework sequence analysis (e.g., as described in Knappik et al, (2000) J Mol Biol 296:57-86), or synthetic HCDR3 bound to a library of human immunoglobulin genes displayed on phage (e.g., as described in Shi et al, (2010) J Mol Biol 397:385-96 and international patent publication No. WO 2009/085462). The definition of "human antibody" excludes antibodies in which at least one CDR is derived from a non-human species.

"humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from a human immunoglobulin sequence. Humanized antibodies may contain substitutions in the framework such that the framework may not be an exact copy of the expressed human immunoglobulin or human immunoglobulin germline gene sequence.

By "in combination with … …" is meant that two or more therapeutic agents are administered to a subject together as a mixture, simultaneously as a single agent, or sequentially in any order as a single agent.

An "intracellular signaling domain" or "cytoplasmic signaling domain" refers to the intracellular portion of a molecule. The functional part of the protein acts by transmitting information within the cell to modulate cellular activity via defined signaling pathways by generating second messengers, or by acting as effectors in response to such messengers. The intracellular signaling domain produces a signal that promotes immune effector function of a CAR-containing cell (e.g., CAR-T cell).

"isolated" refers to a homogeneous population of molecules (such as synthetic polynucleotides or polypeptides) that have been substantially isolated and/or purified from other components of a system that produces the molecules (such as recombinant cells), as well as proteins that have been subjected to at least one purification or isolation step. "isolated" refers to a molecule that is substantially free of other cellular material and/or chemicals, and encompasses molecules that are isolated to a higher purity (such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity).

"modulation" refers to enhancement or reduction of a test molecule mediated by a test molecule when compared to a response mediated by a control or vehicle Response to a requestEnhanced or reduced capability (i.e., downstream effects).

"monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibody molecules, i.e., the individual antibodies comprising the population are identical, except for possible well-known alterations such as removal of C-terminal lysines from the antibody heavy chain or post-translational modifications such as amino acid isomerisation or deamidation, methionine oxidation or asparagine or glutamine deamidation. Monoclonal antibodies typically bind to an epitope. Bispecific monoclonal antibodies bind two different epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibodies may be monospecific or multispecific, such as bispecific, monovalent, bivalent or multivalent.

"multispecific" refers to a molecule, such as an antibody, that specifically binds to two or more different antigens or two or more different epitopes within the same antigen. The multispecific molecule may be cross-reactive to other related antigens, for example, to the same antigen from other species (homologous), such as humans or monkeys, e.g., cynomolgus monkey (cynomolgus) or chimpanzee, or may bind an epitope shared between two or more different antigens.

"Natural killer cells" and "NK cells" are used interchangeably herein and are synonymously used herein. NK cells are referred to as having CD16 ⁺ CD56 ⁺ And/or CD57 ⁺ TCR ^- Phenotypic differentiated lymphocytes. NK cells are characterized by their ability to bind and kill cells that are not expressing "self" MHC/HLA antigens by activating specific cytolysins, their ability to kill tumor cells or other diseased cells that express ligands for NK-activated receptors, and their ability to release protein molecules known as cytokines that stimulate or suppress immune responses.

"operative linkage" and like phrases, when used with respect to nucleic acids or amino acids, refer to the operative linkage of nucleic acid sequences or amino acid sequences, respectively, that are in functional relationship to one another. For example, operably linked promoters, enhancer elements, open reading frames, 5 'and 3' utr, and terminator sequences result in the accurate production of nucleic acid molecules (e.g., RNA), and in some cases, the production of polypeptides (i.e., expression of open reading frames). An operatively linked peptide refers to a peptide in which the functional domains of the peptide are placed at an appropriate distance from each other to confer the intended function of each domain.

The term "paratope" refers to a region or region of an antibody molecule that is involved in antigen binding and that comprises residues that interact with an antigen. The paratope may be comprised of contiguous and/or non-contiguous amino acids that form conformational space units. The paratope of a given antibody can be defined and characterized at different levels of detail using various experimental and computational methods. Experimental methods include hydrogen/deuterium exchange mass spectrometry (HX-MS). Complementary bits will be defined differently depending on the positioning method employed.

"pharmaceutical composition" refers to a combination of two or more active ingredients administered together or separately.

"pharmaceutical composition" refers to a composition of an active ingredient in combination with a pharmaceutically acceptable carrier.

By "pharmaceutically acceptable carrier" or "excipient" is meant an ingredient in a pharmaceutical composition other than the active ingredient, which is non-toxic to the subject. Exemplary pharmaceutically acceptable carriers are buffers, stabilizers or preservatives.

"Polynucleotide" or "nucleic acid" refers to a synthetic molecule comprising nucleotide chains or other equivalent covalent chemicals covalently linked to a phosphosaccharide backbone. cDNA is a typical example of a polynucleotide. The polynucleotide may be a DNA or RNA molecule.

By "preventing" a disease or disorder is meant preventing the occurrence of the disorder in a subject.

"proliferation" refers to an increase in cell division (symmetrical or asymmetrical division of cells).

"promoter" refers to the smallest sequence required to initiate transcription. Promoters may also include enhancer or repressor elements that enhance or repress transcription, respectively.

"protein" or "polypeptide" is used interchangeably herein and refers to a molecule comprising one or more polypeptides, each comprising at least two amino acid residues linked by peptide bonds. The protein may be a monomer or may be a protein complex of two or more subunits, the subunits being the same or different. Small polypeptides of less than 50 amino acids may be referred to as "peptides". The protein may be a heterologous fusion protein, glycoprotein or a protein modified by post-translational modifications such as phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutarition, ADP-ribosylation, pegylation or biotinylation. The protein may be an antibody or may comprise an antigen binding fragment of an antibody. The protein may be recombinantly expressed.

"prostate specific membrane antigen" or "PSMA" refers to a type II membrane protein expressed on certain cells. The amino acid sequence of human PSMA is shown as SEQ ID NO. 240. The extracellular domain spans residues 44-750 of SEQ ID NO. 240, the transmembrane domain spans residues 20-43 of SEQ ID NO. 240, and the cytoplasmic domain spans residues 1-19 of SEQ ID NO. 240.

SEQ ID NO:240

MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIY APSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA

"recombinant" refers to polynucleotides, polypeptides, vectors, viruses, and other macromolecules that are prepared, expressed, created, or isolated by recombinant means.

"regulatory element" refers to any cis-or trans-acting gene element that controls some aspect of the expression of a nucleic acid sequence.

"recurrence" refers to the amelioration of the disease or the recurrence of signs and symptoms of the disease after a period of time following prior treatment with a therapeutic agent.

"refractory" refers to a disease that does not respond to treatment. The refractory disease may be resistant to treatment prior to or at the beginning of treatment, or the refractory disease may become resistant during treatment.

"Single chain Fv" or "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region (VL) and at least one antibody fragment comprising a heavy chain variable region (VH), wherein the VL and VH are linked consecutively via a polypeptide linker and are capable of being expressed as a single chain polypeptide. As used herein, an scFv may have a VL variable region and a VH variable region in either order, e.g., the scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL, relative to the N-terminus and the C-terminus of the polypeptide, unless otherwise indicated.

“(scFv) ₂ "or" tandem scFv "or" double scFv "fragment refers to a fusion protein comprising two light chain variable regions (VLs) and two heavy chain variable regions (VH), wherein the two VLs and the two VH are connected in series via a polypeptide linker and are capable of being expressed as a single chain polypeptide. The two VLs and the two VH are fused by a peptide linker to form a bivalent molecule VL _A linker-VH _A linker-VL _B linker-VH _B To form two binding sites capable of simultaneously binding two different antigens or epitopes.

"specific binding" or "binding" refers to the binding of a proteinaceous molecule to an antigen or epitope within an antigen with greater affinity than to other antigens. Typically, protein molecules bind to an antigen or epitope within an antigen, equilibrium dissociation constants (K _D ) Is about 1X 10 ^-7 M or less, e.g. about 5X 10 ^-8 M or less, about 1X 10 ^-8 M or less, about 1X 10 ^-9 M or less, about 1X 10 ^-10 M or less, about 1X 10 ^-11 M or less or about 1X 10 ^-12 M or less, usually K _D K in comparison to its binding to non-specific antigens (e.g. BSA, casein) _D At least one hundred times lower. In the context of the prostate neoantigen described herein, "specific binding" refers to binding of a proteinaceous molecule to the prostate neoantigen, but not to detectable binding to the wild-type protein of which the neoantigen is a variant.

"subject" includes any human or non-human animal. "non-human animals" include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like. The terms "subject" and "patient" are used interchangeably herein.

"T cells" and "T lymphocytes" are interchangeable herein and are used synonymously herein. T cells include thymic cells, naive T lymphocytes, memory T cells, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes. The T cell may be a T helper (Th) cell, such as a T helper 1 (Th 1) or T helper 2 (Th 2) cell. T cells may be helper T cells (HTL; CD 4) ⁺ T cells, CD4 ⁺ T cells, cytotoxic T cells (CTL; CD 8) ⁺ T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8 ⁺ T cells, CD4 ⁺ CD8 ⁺ T cells or any other subpopulation of T cells. Also included are "NKT cells" which refer to a specialized T cell population that expresses a semi-invariant αβ T cell receptor but also expresses a variety of molecular markers commonly associated with NK cells (such as NK 1.1). NKT cells include NK1.1 ⁺ Cells and NK1.1 ^- Cells and CD4 ⁺ Cell, CD4 ^- Cell, CD8 ⁺ Cell and CD8 ^- And (3) cells. The TCR on NKT cells is unique in that it recognizes a glycolipid antigen presented by the MHC I-like molecule CD Id. NKT cells may have protective or deleterious effects, as they are capable of producing cytokines that promote inflammation or immune tolerance. Also included are "gamma-delta T cells" (γδ T cells) which refer to a specialized population, i.e., a small subset of T cells having a unique TCR on their surface, and unlike most T cells in which the TCR consists of two glycoprotein chains designated as the α -TCR chain and the β -TCR chain, the TCR in γδ T cells consists of the γ -chain and the δ -chain. γδ T cells can play a role in immune surveillance and immunomodulation, and have been found to be an important source of IL-17 and induce strong CD8 ⁺ Cytotoxic T cell response. Also included are "regulatory T cells" or "tregs," which refer to T cells that suppress abnormal or excessive immune responses and play a role in immune tolerance. Treg is usually the transcription factor Foxp 3-positive CD4 ⁺ T cells, and may also include transcription factor Foxp 3-negative regulatory T cells, which are IL-10 producing CD4 ⁺ T cells.

"therapeutically effective amount" or "effective amount" is used interchangeably herein to refer to an amount effective to achieve a desired therapeutic result at a desired dosage and for a desired period of time. The therapeutically effective amount may vary depending on the following factors: such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example: improvement of patient health, reduction of tumor burden, inhibition or slowing of tumor growth, and/or the absence of metastasis of cancer cells to other parts of the body.

"transduction" refers to the use of viral vectors to introduce foreign nucleic acids into cells.

"treating" of a disease or disorder, such as cancer, refers to effecting one or more of the following: reducing the severity and/or duration of the disorder, inhibiting exacerbation of symptoms characteristic of the disorder being treated, limiting or preventing recurrence of the disorder in a subject previously suffering from the disorder, or limiting or preventing recurrence of the symptom in a subject previously suffering from the symptom of the disorder.

"tumor cell" or "cancer cell" refers to a cancerous, precancerous, or transformed cell in vivo, ex vivo, or in tissue culture, which has a spontaneous or induced phenotypic change. These changes do not necessarily involve uptake of new genetic material. Transformation may occur by infection of the transformed virus, as well as binding to new genomic nucleic acid, uptake of exogenous nucleic acid, or it may occur spontaneously or after exposure to a carcinogen, thereby mutating the endogenous gene. Transformation/cancer is exemplified by morphological changes in vitro, in vivo and ex vivo, cell immortality, abnormal growth control, lesion formation, proliferation, malignancy, tumor-specific marker level modulation, invasion, tumor growth in a suitable animal host (such as nude mice, etc.).

"variant," "mutant," or "altered" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide by one or more modifications (e.g., one or more substitutions, insertions, or deletions).

Throughout the specification, numbering of amino acid residues in the constant region of an antibody is performed according to the EU index as described in Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, MD. (1991), unless explicitly stated otherwise.

Mutations in the Ig constant region are referred to as follows: l351y_f405a_y407V refers to the L351Y, F a and Y407V mutations in one immunoglobulin constant region. L351Y_F405A_Y407V/T394W refers to the L351Y, F405A and Y407V mutations in the first Ig constant region and the T394W mutation in the second Ig constant region present in a multimeric protein.

"VHH" refers to a single domain antibody or nanobody that consists of only heavy chain homodimers. VHH single domain antibodies lack the light and heavy chain CH1 domains of the conventional Fab region.

Unless otherwise indicated, any numerical values, such as concentrations or ranges of concentrations described herein, are to be understood as being modified in all instances by the term "about. Thus, a numerical value typically includes ±10% of the value. For example, a concentration of 1mg/mL includes 0.9mg/mL to 1.1mg/mL. Also, the concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, a numerical range, unless the context clearly indicates otherwise, includes all possible subranges, all individual values within the range, including integers within such range and fractions within the range.

TABLE 1 conventional single and three letter amino acid codes used herein

Amino acids	Three letter code	Single letter code
			Alanine (Ala)	Ala	A
Arginine (Arg)	Arg	R
			Asparagine derivatives	Asn	N
Aspartic acid	Asp	D
			Cysteine (S)	Cys	C
Glutamic acid	Glu	E
			Glutamine	Gln	Q
Glycine (Gly)	Gly	G
			Histidine	His	H
Isoleucine (Ile)	Ile	I
			Lysine	Lys	K
Methionine	Met	M
			Phenylalanine (Phe)	Phe	F
Proline (proline)	Pro	P
			Serine (serine)	Ser	S
Threonine (Thr)	Thr	T
			Tryptophan	Trp	W
Tyrosine	Tyr	Y
			Valine (valine)	Val	V

Antigen binding domains that bind CD3 epsilon。

The present disclosure provides antigen binding domains that bind CD3 epsilon, monospecific and multispecific proteins comprising antigen binding domains that bind CD3 epsilon, polynucleotides encoding the foregoing, vectors, host cells, methods of making and using the foregoing. The antigen binding domains identified herein that bind CD3 epsilon demonstrate several advantageous properties. First, IGHV1-69 x 02-IGHJ1-01 and IGKV3-11 x 02-IGKJ4-01 lines were selected for CDR grafting to ensure enhanced binding compared to the murine Cris-7 parent antibody. Second, after introduction of human-mouse mutations, selected clones exhibit improved thermostability by retaining binding after heat shock (including at 55 ℃, 60 ℃ and/or 65 ℃), a feature that leads to improved manufacturability and storage. This is not the case for the murine Cris-7 parent antibody, which exhibits only minimal binding to recombinant CD3 cells and T cells after heat shock when compared to the antigen binding domain of the invention that binds CD3 epsilon. Third, by making a substitution at position N106 in SEQ ID NOS: 55, 54 and 48, thereby preventing Asn deamidation, the risk of post-translational modification (PTM) is lessened, whereas if Asn deamidation is not modified, loss of activity may result. The engineered position at residue N106 is within HCDR 3. Even with mutations at this location within HCR3, antibodies remain capable of binding antigen robustly while possessing additional beneficial properties (e.g., improved thermostability).

The present disclosure also provides an isolated protein comprising an antigen binding domain that binds CD3 epsilon, wherein the antigen binding domain that binds CD3 epsilon comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 86, 79, 80 and 81, respectively. SEQ ID NO. 86 (PQVHYDYXGFPY, where X may be Q, A, G or S) represents a broad class of HCDR3 amino acid sequences comprising variants exhibiting improved properties including improved thermostability, reduced risk of deamidation, and differences in affinity for CD3 depending on the amino acid substituted for "X". For example, if X in SEQ ID NO:86 is substituted with Q or A, then CD3 affinity is similar to that of the parent (X is replaced with N); if X is substituted with G or S, then CD3 affinity is lower than Q or A. This provides the advantageous ability to modulate T cell redirecting ability activity of a multi or bispecific protein comprising a CD3 epsilon binding domain of the disclosure, in order to potentially reduce cytokine responses in a subject and potentially enhance tumor distribution of the multi or bispecific protein.

The present disclosure provides an isolated protein comprising an antigen binding domain that binds CD3 epsilon, wherein the antigen binding domain that binds CD3 epsilon comprises:

HCDR1, HCDR2 and HCDR3 of the heavy chain variable region (VH) of SEQ ID No. 55, and light chain complementarity determining region (LCDR) 1, LCDR2 and LCDR3 of the light chain variable region (VL) of SEQ ID No. 59;

The present disclosure provides an isolated protein comprising an antigen binding domain that binds CD3 epsilon, wherein the antigen binding domain that binds CD3 epsilon comprises HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 86, 79, 80 and 81, respectively.

The present disclosure provides an isolated protein comprising an antigen binding domain that binds CD3 epsilon, wherein the antigen binding domain that binds CD3 epsilon comprises HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 as follows:

SEQ ID NOs 70, 71, 72, 79, 80 and 81, respectively;

70, 71, 87, 79, 80 and 81 respectively; or alternatively

SEQ ID NOS 70, 71, 90, 79, 80 and 81, respectively.

The present disclosure provides an isolated protein comprising an antigen binding domain that binds CD3 epsilon, wherein the antigen binding domain that binds CD3 epsilon comprises the VH of SEQ ID NO:55, 54 or 48, and the VL of SEQ ID NO:59, 58 or 56.

VH of SEQ ID NO. 55 and VL of SEQ ID NO. 59;

VH of SEQ ID NO. 55 and VL of SEQ ID NO. 58;

VH of SEQ ID NO. 54 and VL of SEQ ID NO. 56; or alternatively

VH of SEQ ID NO. 58 and VL of SEQ ID NO. 58.

In other embodiments, the antigen binding domain that binds CD3 epsilon is a (scFv) ₂ 。

In other embodiments, the antigen binding domain that binds CD3 epsilon is an Fv.

In other embodiments, the antigen binding domain that binds CD3 ε is F (ab') ₂ 。

In other embodiments, the antigen binding domain that binds CD3 epsilon is Fd.

In other embodiments, the CD3 epsilon antigen binding domain is a dAb.

In other embodiments, the CD3 epsilon antigen binding domain is a VHH.

scFv binding CD3 epsilon

Either of the VH domain and VL domain identified herein that bind CD3 epsilon may be engineered into VH-linker-VL or VL-linker-VH oriented scFv formats. Any of the VH domains and VL domains identified herein may also be used to generate sc (Fv) ₂ Structures such as VH-linker-VL-linker-VH, VH-linker-VL-linker-VH-linker-VL. VH-linker-VL. VL-linker-VH-linker-VL. VL-linker-VH-linker-VL-linker-VH or VL-linker-VH.

The VH and VL domains identified herein may be incorporated into scFv formats, and the resulting scFv may be assessed for binding to CD3 epsilon and thermostability using known methods. Binding can be assessed using a Proteon XPR36, biacore 3000 or KinExA instrument, ELISA or competitive binding assay known to those skilled in the art. Purified scFv or e.coli supernatant or lysed cells containing expressed scFv can be used to assess binding. The measured affinity of the test scFv for CD3 epsilon can vary if measured under different conditions (e.g., osmolarity, pH). Thus, measurements of affinity and other binding parameters (e.g., KD, kon, and Koff) are typically made with standardized conditions and standardized buffers. Thermal stability can be assessed by heating the test scFv at an elevated temperature (such as 50 ℃, 55 ℃, or 60 ℃) for a period of time (such as 5 minutes (min), 10min, 15min, 20min, 25min, or 30 min) and measuring binding of the test scFv to CD3 epsilon. The scFv remained equally bound to CD3 epsilon when compared to the unheated scFv sample, referred to as thermostable.

In recombinant expression systems, the linker is a peptide linker and may comprise any naturally occurring amino acid. Exemplary amino acids that may be included in The linker are Gly, ser, pro, thr, glu, lys, arg, ile, leu, his and The. The length of the linker should be sufficient to allow the VH and VL to be linked in the correct conformation relative to each other so that they retain the desired activity (such as binding to CD3 epsilon).

The length of the linker may be about 5 to 50 amino acids. In other embodiments, the linker is about 10 to 40 amino acids in length. In other embodiments, the linker is about 10 to 35 amino acids in length. In other embodiments, the linker is about 10 to 30 amino acids in length. In other embodiments, the linker is about 10 to 25 amino acids in length. In other embodiments, the linker is about 10 to 20 amino acids in length. In other embodiments, the linker is about 15 to 20 amino acids in length. In other embodiments, the linker is about 16 to 19 amino acids in length. In other embodiments, the linker is 6 amino acids in length. In other embodiments, the linker is 7 amino acids in length. In other embodiments, the linker is 8 amino acids in length. In other embodiments, the linker is 9 amino acids in length. In other embodiments, the linker is 10 amino acids in length. In other embodiments, the linker is 11 amino acids in length. In other embodiments, the linker is 12 amino acids in length. In other embodiments, the linker is 13 amino acids in length. In other embodiments, the linker is 14 amino acids in length. In other embodiments, the linker is 15 amino acids in length. In other embodiments, the linker is 16 amino acids in length. In other embodiments, the linker is 17 amino acids in length. In other embodiments, the linker is 18 amino acids in length. In other embodiments, the linker is 19 amino acids in length. In other embodiments, the linker is 20 amino acids in length. In other embodiments, the linker is 21 amino acids in length. In other embodiments, the linker is 22 amino acids in length. In other embodiments, the linker is 23 amino acids in length. In other embodiments, the linker is 24 amino acids in length. In other embodiments, the linker is 25 amino acids in length. In other embodiments, the linker is 26 amino acids in length. In other embodiments, the linker is 27 amino acids in length. In other embodiments, the linker is 28 amino acids in length. In other embodiments, the linker is 29 amino acids in length. In other embodiments, the linker is 30 amino acids in length. In other embodiments, the linker is 31 amino acids in length. In other embodiments, the linker is 32 amino acids in length. In other embodiments, the linker is 33 amino acids in length. In other embodiments, the linker is 34 amino acids in length. In other embodiments, the linker is 35 amino acids in length. In other embodiments, the linker is 36 amino acids in length. In other embodiments, the linker is 37 amino acids in length. In other embodiments, the linker is 38 amino acids in length. In other embodiments, the linker is 39 amino acids in length. In other embodiments, the linker is 40 amino acids in length. Exemplary linkers that can be used are Gly-rich linkers, gly-and Ser-containing linkers, gly-and Ala-containing linkers, ala-and Ser-containing linkers, and other flexible linkers.

Other linker sequences may include portions of immunoglobulin hinge regions, CL or CH1 derived from any immunoglobulin heavy or light chain isotype. Alternatively, a variety of non-protein polymers, including polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol may be used as linkers. Exemplary joints that may be used are shown in table 2. Additional linkers are described, for example, in International patent publication WO 2019/060695.

TABLE 2 Joint

In other embodiments, the scFv comprises, from N-terminus to C-terminus, a VH, a first linker (L1), and a VL (VH-L1-VL).

In other embodiments, the scFv comprises from N-terminus to C-terminus a VL, L1 and VH (VL-L1-VH).

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 3.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 4.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 5.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 6.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 7.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 8.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 9.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 10.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 11.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 12.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 13.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 14.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 15.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 16.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 17.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 19.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 20.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 21.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 22.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 23.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 24.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 25.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 26.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 27.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 28.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 29.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 30.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 31.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 32.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 33.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 34.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 35.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 36.

In other embodiments, the scFv comprises:

In other embodiments, the scFv comprises HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 86, 79, 80 and 81, respectively.

In other embodiments, the scFv comprises HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 as follows:

SEQ ID NOs 70, 71, 72, 79, 80 and 81, respectively;

70, 71, 87, 79, 80 and 81 respectively; or alternatively

SEQ ID NOS 70, 71, 90, 79, 80 and 81, respectively.

In other embodiments, the scFv comprises HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 72, 79, 80 and 81, respectively.

In other embodiments, the scFv comprises HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 87, 79, 80 and 81, respectively.

In other embodiments, the scFv comprises HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 90, 79, 80 and 81, respectively.

In other embodiments, the scFv comprises the VH of SEQ ID NO:55, 54 or 48 and the VL of SEQ ID NO:59, 58 or 56.

In other embodiments, the scFv comprises the VH of SEQ ID NO:55 and the VL of SEQ ID NO: 59.

In other embodiments, the scFv comprises the VH of SEQ ID NO:55 and the VL of SEQ ID NO: 58.

In other embodiments, the scFv comprises the VH of SEQ ID NO:54 and the VL of SEQ ID NO: 56.

In other embodiments, the scFv comprises the VH of SEQ ID NO. 48 and the VL of SEQ ID NO. 58.

In other embodiments, the scFv comprises the VH of SEQ ID NO. 88 and the VL of SEQ ID NO. 58.

In other embodiments, the scFv comprises the VH of SEQ ID NO:242 and the VL of SEQ ID NO: 58.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125 or 126.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 96.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 97.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 98.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 99.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 100.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 101.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 102.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 103.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 104.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 105.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 106.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 107.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 108.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 109.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 110.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 111.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 112.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 113.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 114.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 115.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 116.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 117.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 118.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 119.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 120.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 121.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 122.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 123.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 124.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 125.

In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 126.

Other antigen binding domains that bind CD3 epsilon

Any of the VH and VL domains identified herein that bind CD3 epsilon can also be engineered into Fab, F (ab') 2, fd, or Fv formats and their binding to CD3 epsilon and thermostability can be assessed using the assays described herein. In certain embodiments, the thermostability at 55 ℃, 60 ℃ and/or 65 ℃ is increased by a factor of 2, 3, 4, 5, up to 100, and each integer multiple (e.g., 6, 7, 8, 9, 10, etc.) between these multiples as compared to the murine Cris-7 parent antibody by the methods described herein.

In other embodiments, the Fab comprises:

In other embodiments, the Fab comprises HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 86, 79, 80 and 81, respectively.

In other embodiments, the Fab comprises the following sequences HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3:

70, 71, 72, 79, 80 and 81;

70, 71, 87, 79, 80 and 81; or alternatively

SEQ ID NOS 70, 71, 90, 79, 80 and 81.

In other embodiments, the Fab comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 72, 79, 80 and 81, respectively.

In other embodiments, the Fab comprises HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 87, 79, 80 and 81, respectively.

In other embodiments, the Fab comprises HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 90, 79, 80 and 81, respectively.

In other embodiments, the Fab comprises the VH of SEQ ID NO. 55 and the VL of SEQ ID NO. 59.

In other embodiments, the Fab comprises the VH of SEQ ID NO:55 and the VL of SEQ ID NO: 58.

In other embodiments, the Fab comprises the VH of SEQ ID NO. 54 and the VL of SEQ ID NO. 56.

In other embodiments, the Fab comprises the VH of SEQ ID NO. 48 and the VL of SEQ ID NO. 58.

In other embodiments, the Fab comprises the VH of SEQ ID NO. 88 and the VL of SEQ ID NO. 58.

In other embodiments, the Fab comprises the VH of SEQ ID NO:242 and the VL of SEQ ID NO: 58.

In other embodiments, the F (ab') ₂ Comprising:

In other embodiments, the F (ab') ₂ Comprising HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 86, 79, 80 and 81, respectively.

In other embodiments, the F (ab') ₂ HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the following sequences:

70, 71, 72, 79, 80 and 81;

70, 71, 87, 79, 80 and 81; or alternatively

SEQ ID NOS 70, 71, 90, 79, 80 and 81.

In some embodiments, the F (ab') ₂ Comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 72, 79, 80 and 81, respectively.

In other embodiments, the F (ab') ₂ Comprising HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 87, 79, 80 and 81, respectively.

In other embodiments, the F (ab') ₂ Comprising HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 90, 79, 80 and 81, respectively.

In other embodiments, the F (ab') ₂ Comprising the VH of SEQ ID NO. 55 and the VL of SEQ ID NO. 59.

In other embodiments, the F (ab') ₂ Comprising the VH of SEQ ID NO. 55 and the VL of SEQ ID NO. 58.

In other embodiments, the F (ab') ₂ Comprising the VH of SEQ ID NO:54 and the VL of SEQ ID NO: 56.

In other embodiments, the F (ab') ₂ Comprising the VH of SEQ ID NO. 48 and the VL of SEQ ID NO. 58.

In other embodiments, the F (ab') ₂ Comprising the VH of SEQ ID NO. 88 and the VL of SEQ ID NO. 58.

In other embodiments, the F (ab') ₂ Comprising the VH of SEQ ID NO:242 and the VL of SEQ ID NO: 58.

In other embodiments, the Fv comprises:

In other embodiments, the Fv comprises HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 86, 79, 80 and 81, respectively.

In other embodiments, the Fv comprises HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 of the following sequences:

70, 71, 72, 79, 80 and 81;

70, 71, 87, 79, 80 and 81; or alternatively

SEQ ID NOS 70, 71, 90, 79, 80 and 81.

In other embodiments, the Fv comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 72, 79, 80 and 81, respectively.

In other embodiments, the Fv comprises HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 87, 79, 80 and 81, respectively.

In other embodiments, the Fv comprises HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 90, 79, 80 and 81, respectively.

In other embodiments, the Fv comprises the VH of SEQ ID NO. 55 and the VL of SEQ ID NO. 59.

In other embodiments, the Fv comprises the VH of SEQ ID NO. 55 and the VL of SEQ ID NO. 58.

In other embodiments, the Fv comprises the VH of SEQ ID NO. 54 and the VL of SEQ ID NO. 56.

In other embodiments, the Fv comprises the VH of SEQ ID NO. 48 and the VL of SEQ ID NO. 58.

In other embodiments, the Fv comprises the VH of SEQ ID NO. 88 and the VL of SEQ ID NO. 58.

In other embodiments, the Fv comprises the VH of SEQ ID NO. 242 and the VL of SEQ ID NO. 58.

In other embodiments, the Fd comprises:

heavy chain complementarity determining regions (HCDR) 1, HCDR2 and HCDR3 of the heavy chain variable regions (VH) of SEQ ID NOS 55, 54 or 48.

In other embodiments, the Fd comprises HCDR1, HCDR2 and HCDR3 of SEQ ID NOS 70, 71 and 86, respectively.

In other embodiments, the Fd comprises HCDR1, HCDR1 and HCDR3 of SEQ ID NOS 70, 71 and 72, respectively.

In other embodiments, the Fd comprises HCDR1, HCDR1 and HCDR3 of SEQ ID NOS 70, 71 and 87, respectively.

In other embodiments, the Fd comprises HCDR1, HCDR1 and HCDR3 of SEQ ID NOs 70, 71 and 90, respectively.

In other embodiments, the Fd comprises the VH of SEQ ID NO. 55.

In other embodiments, the Fd comprises the VH of SEQ ID NO. 54.

In other embodiments, the Fd comprises the VH of SEQ ID NO. 48.

In other embodiments, the Fd comprises the VH of SEQ ID NO. 88.

In other embodiments, the Fd comprises the VH of SEQ ID NO: 242.

Homologous antigen binding domains and antigen binding domains with conservative substitutions

Variants of the antigen binding domain that bind CD3 epsilon are within the scope of the present disclosure. For example, variants may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acid substitutions in the antigen binding domain that binds CD3 epsilon, so long as they retain or have improved functional properties compared to the parent antigen binding domain. In other embodiments, the sequence identity to the antigen binding domain of the disclosure that binds CD3 epsilon may be about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In other embodiments, the variation is in the framework region. In other embodiments, the variants are generated by conservative substitutions.

For example, an antigen binding domain that binds CD3 epsilon may comprise a substitution at residue position N106 (residue numbering starting from the N-terminus of SEQ ID NO:55, 54 or 48). Conservative substitutions may be made at any given position and the resulting variant antigen binding domain that binds CD3 epsilon is tested for its desired characteristics in the assays described herein.

Also provided is an antigen binding domain that binds CD3 epsilon comprising VH and VL that are at least 80% identical to VH and VL:

VH of SEQ ID NO. 55 and VL of SEQ ID NO. 59;

VH of SEQ ID NO. 55 and VL of SEQ ID NO. 58;

VH of SEQ ID NO. 54 and VL of SEQ ID NO. 56;

VH of SEQ ID NO. 48 and VL of SEQ ID NO. 58;

the VH of SEQ ID NO. 88 and the VL of SEQ ID NO. 58; or alternatively

The VH of SEQ ID NO. 242 and the VL of SEQ ID NO. 58.

In other embodiments, the identity is 85%. In other embodiments, the identity is 90%. In other embodiments, the identity is 91%. In other embodiments, the identity is 91%. In other embodiments, the identity is 92%. In other embodiments, the identity is 93%. In other embodiments, the identity is 94%. In other embodiments, the identity is 94%. In other embodiments, the identity is 95%. In other embodiments, the identity is 96%. In other embodiments, the identity is 97%. In other embodiments, the identity is 98%. In other embodiments, the identity is 99%.

The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., identity% = number of identical positions/total number of positions x 100), and these parameters need to be introduced for optimal alignment of the two sequences taking into account the number of gaps and the length of each gap.

The percent identity between two amino acid sequences can be determined using the algorithm of E.Meyers and W.Miller (Comput Appl Biosci 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weighted residue table, gap length penalty 12 and gap penalty 4. In addition, the percent identity between two amino acid sequences can be determined using Needleman and Wunsch (J Mol Biol 48:444-453 (1970)) algorithms that have been incorporated into the GAP program of the GCG software package (retrievable from the internet < URL: http:// www.gcg.com >), using the blosum 62 matrix or PAM250 matrix, and a GAP weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6.

In other embodiments, the variant antigen-binding domain that binds CD3 epsilon comprises one or two conservative substitutions in either of the CDR regions, while maintaining the desired functional properties of the parent antigen-binding fragment that binds CD3 epsilon.

By "conservative modification" is meant an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody that contains the amino acid modification. Conservative modifications include amino acid substitutions, additions, and deletions. A "conservative amino acid substitution" is a substitution in which an amino acid is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are well-defined and include amino acids with the following side chains: acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine) and sulfur-containing side chains (cysteine, methionine). In addition, any of the natural residues in the polypeptide may also be substituted with alanine as described previously for alanine scanning mutagenesis (MacLennan et al, (1988) Acta Physiol Scand Suppl 643:55-67; sasaki et al, (1988) Adv Biophys 35:1-24). Amino acid substitutions to the antibodies of the invention may be made by known methods, such as by PCR mutagenesis (U.S. Pat. No. 4,683,195). Alternatively, a library of variants may be generated, for example, using random (NNK) or non-random codons, e.g., DVK codons, which encode 11 amino acids (Ala, cys, asp, glu, gly, lys, asn, arg, ser, tyr, trp). The resulting variants can be tested for characteristics using the assays described herein.

Method of generating antigen binding fragments that bind CD3 epsilon

Various techniques can be used to generate the antigen binding domains provided in the present disclosure that bind CD3 epsilon. For example, the hybridoma method of Kohler and Milstein can be used to identify VH/VL pairs that bind CD3 epsilon. In the hybridoma method, a mouse or other host animal (such as hamster, rat, or chicken) is immunized with human and/or cyno CD3 epsilon, and spleen cells from the immunized animal are then fused with myeloma cells to form hybridoma cells using standard methods. Colonies generated by individual immortalized hybridoma cells can be screened for the production of antibodies containing an antigen binding domain that binds CD3 epsilon with desired characteristics, such as binding specificity, cross-reactivity or lack of binding specificity, lack of cross-reactivity, affinity for antigen, and any desired functionality.

The antigen binding domain that binds CD3 epsilon generated by immunization of a non-human animal may be humanized. Exemplary humanization techniques including selection of human acceptor frameworks include CDR grafting (U.S. Pat. No. 5,225,539), SDR grafting (U.S. Pat. No. 6,818,749), surface remodeling (Padlan, (1991) Mol Immunol 28:489-499), specificity determining residue surface remodeling (U.S. patent publication 2010/0261620), human framework remodelling (U.S. Pat. 8,748,356), or superhumanization (U.S. Pat. No. 7,709,226). In these methods, the CDRs or a subset of CDR residues of the parent antibody are transferred to a human framework, which can be selected based on its overall homology to the parent framework, based on similarity of CDR lengths or canonical structural identity, or a combination thereof.

The humanized antigen binding domain can be further optimized to improve its selectivity or affinity for the desired antigen by: the binding affinity is maintained by incorporating altered framework support residues (back mutations) using techniques such as those described in International patent publication Nos. WO1090/007861 and WO1992/22653, or the affinity of the antigen binding domain is improved by introducing variations at any CDR, for example.

Transgenic animals (such as mice, rats or chickens) carrying human immunoglobulin (Ig) loci in their genomes can be used to generate antigen binding fragments that bind CD3 epsilon and are described, for example, in U.S. Pat. No. 6,150,584, international patent publication No. WO1999/45962, international patent publications nos. WO2002/066630, WO2002/43478, WO2002/043478, and WO 1990/04036. Endogenous immunoglobulin loci in such animals may be disrupted or deleted and at least one full or partial human immunoglobulin locus may be inserted into the genome of the animal by homologous or nonhomologous recombination using a transchromosome or minigene. Companies such as Regeneron (< URL: http:// www.regeneron.com >), harbour Antibodies (http:// www.harbourantibodies.com), open Monoclonal Technology, inc. (OMT) (< URL: http:// www.omtinc.net >), kyMab (< URL: http:// www.kymab.com >), trianni (< URL: http:// www.trianni.com >) and Ablexis (< URL: http:// www.ablexis.com >) can be invited to use the techniques described above to provide human antibodies to the selected antigen.

The antigen binding domain that binds CD3 epsilon may be selected from phage display libraries in which phage are engineered to express human immunoglobulins or portions thereof, such as Fab, single chain antibodies (scFv), or unpaired or paired antibody variable regions. The antigen binding domain that binds CD3 epsilon can be isolated, for example, from a phage display library expressing the antibody heavy and light chain variable regions as fusion proteins using phage pIX coat proteins, as described in Shi et al, (2010) J Mol Biol 397:385-96 and International patent publication No. WO 09/085462. Phage binding to human and/or cyno CD3 epsilon can be screened from the library and the positive clones obtained can be further characterized, fab isolated from the clone lysate and converted to scFv or other configurations of antigen binding fragments.

The preparation of the immunogenic antigen and the generation of the antigen binding domains of the present disclosure can be performed using any suitable technique, such as recombinant protein production. The immunogenic antigen can be administered to the animal in the form of a purified protein or protein mixture (including whole cells or cell or tissue extracts), or the antigen can be formed de novo in the animal from nucleic acids encoding the antigen or portions thereof.

Conjugation to half-life extending moieties

The antigen binding domains of the present disclosure that bind CD3 epsilon can be conjugated to a half-life extending moiety. Exemplary half-life extending moieties are albumin, albumin variants, albumin binding proteins and/or domains, transferrin and fragments and analogs thereof, immunoglobulins (Ig) or fragments thereof (such as the Fc region). The amino acid sequence of the aforementioned half-life extending moiety is known. Ig or fragments thereof include all isotypes (i.e., igG1, igG2, igG3, igG4, igM, igA, and IgE).

Additional half-life extending moieties that can be conjugated to the CD3 epsilon binding antigen binding domains of the present disclosure include, for example, polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20,000; fatty acids and fatty acid esters of different chain lengths, such as laurate, myristate, stearate, arachidate, behenate, oleate, arachidonic acid, suberic acid, tetradecanedioic acid, octadecanedioic acid, docanedioic acid, etc., polylysine, octane, carbohydrates (dextran, cellulose, oligosaccharides or polysaccharides) in order to obtain the desired properties. These moieties may be fused directly to the CD3 epsilon binding antigen binding domain of the present disclosure and may be generated by standard cloning and expression techniques. Alternatively, well-known chemical coupling methods may be used to attach these moieties to recombinantly produced antigen binding domains of the present disclosure that bind CD3 epsilon.

The polyethylene glycol moiety may be conjugated to the antigen binding domain of the present disclosure that binds CD3 epsilon, for example, by incorporating a cysteine residue into the C-terminus of the antigen binding domain of the present disclosure that binds CD3 epsilon or engineering a cysteine into a residue position that is away from the CD3 epsilon binding site and attaching a polyethylene glycol group to the cysteine using well known methods.

In other embodiments, an antigen binding fragment that binds CD3 epsilon is conjugated to a half-life extending moiety.

In other embodiments, the half-life extending moiety is an immunoglobulin (Ig), a fragment of the Ig, an Ig constant region, a fragment of the Ig constant region, an Fc region, transferrin, albumin, an albumin binding domain, or polyethylene glycol. In other embodiments, the half-life extending moiety is an Ig constant region.

In other embodiments, the half-life extending moiety is Ig.

In other embodiments, the half-life extending moiety is a fragment of Ig.

In other embodiments, the half-life extending moiety is an Ig constant region.

In other embodiments, the half-life extending moiety is a fragment of an Ig constant region.

In other embodiments, the half-life extending moiety is an Fc region.

In other embodiments, the half-life extending moiety is albumin.

In other embodiments, the half-life extending moiety is an albumin binding domain.

In other embodiments, the half-life extending moiety is transferrin.

In other embodiments, the half-life extending moiety is polyethylene glycol.

The pharmacokinetic properties of the CD3 epsilon binding antigen binding domain conjugated to the half-life extending moiety can be evaluated using known in vivo models.

Conjugated to immunoglobulin (Ig) constant regions or fragments of Ig constant regions

The antigen binding domains of the present disclosure that bind CD3 epsilon may be conjugated to Ig constant regions or fragments of Ig constant regions to confer antibody-like properties, including Fc effector function, i.e., C1q binding, complement Dependent Cytotoxicity (CDC), fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis or downregulation of cell surface receptors (e.g., B cell receptors; BCR). The Ig constant region or fragment of the Ig constant region also functions as a half-life extending moiety as discussed herein. The antigen binding domains of the present disclosure that bind CD3 epsilon can be engineered into conventional full length antibodies using standard methods. Full length antibodies comprising an antigen binding domain that binds CD3 epsilon may be further engineered as described herein.

The immunoglobulin heavy chain constant region is composed of subdomains CH1, hinge, CH2, and CH 3. The CH1 domain spans residues A118-V215, CH2 domain residues A231-K340 and CH3 domain residues G341-K447 on the heavy chain, with residue numbering according to the EU index. In some cases, G341 is referred to as a CH2 domain residue. The hinge is generally defined as P230 comprising E216 and ending in human IgG 1. The Ig Fc region comprises at least the CH2 and CH3 domains of the Ig constant region and thus at least the region of the Ig heavy chain constant region about A231 to K447.

The invention also provides an antigen binding domain that binds CD3 epsilon conjugated to an immunoglobulin (Ig) constant region or fragment of an Ig constant region.

In other embodiments, the Ig constant region is a heavy chain constant region.

In other embodiments, the Ig constant region is a light chain constant region.

In other embodiments, a fragment of the Ig constant region comprises at least a portion of a hinge, a CH2 domain, and a CH3 domain. A portion of a hinge refers to one or more amino acid residues of an Ig hinge.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 3.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 4.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 5.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 6.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 7.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 8.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 9.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 10.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 11.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 12.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 13.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 14.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 15.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 16.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 17.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 19.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 20.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 21.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 22.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 23.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 24.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 25.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 26.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 27.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 28.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 29.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 30.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 31.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 32.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 33.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 34.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 35.

In other embodiments, the L2 comprises the amino acid sequence of SEQ ID NO. 36.

Several known assays can be used to evaluate the CD3 epsilon binding antigen binding domains of the present disclosure conjugated to Ig constant regions or fragments of Ig constant regions. Binding to CD3 epsilon can be assessed using the methods described herein. The altered properties conferred by an Ig constant domain or fragment of an Ig constant region (such as an Fc region) can be determined in an Fc receptor binding assay using a soluble form of the receptor, such as fcγri, fcγrii, fcγriii, or FcRn receptor, or using a cell-based assay that measures, for example, ADCC, CDC, or ADCP.

ADCC can be assessed using an in vitro assay using cells expressing CD3 epsilon as target cells and NK cells as effector cells. Cell lysis is detected based on a label (e.g., a radioactive substrate, fluorescent dye, or native intracellular protein) released from the lysed cells. In an exemplary assay, target cells can be used in a ratio of 1 target cell to 4 effector cells. Target cells were pre-labeled with BATDA and mixed with effector cells and test antibodies. The samples were incubated for 2 hours and the cell lysis rate was measured by measuring the BATDA released into the supernatant. Data were normalized to the maximum cytotoxicity using 0.67% Triton X-100 (Sigma Aldrich) and the minimum control determined by the spontaneous release of BATDA from target cells in the absence of any antibodies.

ADCP can be assessed by using monocyte-derived macrophages as effector cells and any CD3 epsilon expressing cells as target cells, which are engineered to express GFP or other marker molecules. In an exemplary assay, the effector cell to target cell ratio can be, for example, 4:1. Effector cells may be incubated with target cells for 4 hours with or without antibodies of the invention. After incubation, cells can be isolated using cell digests (accutase). Macrophages can be identified using anti-CD 11b antibodies and anti-CD 14 antibodies conjugated to fluorescent markers, and CD 11-based using standard methods ⁺ CD14 ⁺ Percent phagocytosis was determined by% GFP fluorescence in macrophages.

CDC of cells can be measured, for example, by: daudi cells were grown at 1X 10 ⁵ Individual cells/well (50 μl/well) were inoculated into RPMI-B (RPMI supplemented with 1% BSA), 50 μl of test protein was added to the well at a final concentration of 0 μg/mL to 100 μg/mL, the reaction was incubated at room temperature for 15min, 11 μl of mixed human serum was added to the well, and then the reaction was incubated at 37 ℃ for 45min. Propidium iodide stained% cells can be detected as percent (%) lysed cells in a FACS assay using standard methods.

Proteins comprising the antigen binding domain of the present disclosure that binds CD3 epsilon

The antigen binding domains of the present disclosure that bind CD3 epsilon can be engineered into various designed monospecific or multispecific proteins using standard methods.

The present disclosure also provides a monospecific protein comprising the antigen binding domain of the disclosure that binds CD3 epsilon.

In other embodiments, the monospecific protein is an antibody.

The present disclosure also provides a multispecific protein comprising an antigen-binding domain of the present disclosure that binds CD3 epsilon.

In other embodiments, the multispecific protein is bispecific.

In other embodiments, the multispecific protein is trispecific.

In other embodiments, the multispecific protein is tetraspecific.

In other embodiments, the multispecific protein is monovalent for binding to CD3 epsilon.

In other embodiments, the multispecific protein is bivalent for binding to CD3 epsilon.

The present disclosure also provides an isolated multi-specific protein comprising a first antigen binding domain that binds CD3 epsilon and a second antigen binding domain that binds a tumor antigen. In other embodiments, the tumor antigen is a protein or fragment thereof that is present on or specific for a cancer cell.

In other embodiments, the tumor antigen is a BCMA antigen. In other embodiments, the tumor antigen is a PSMA antigen. In other embodiments, the tumor antigen is a CD79b antigen. In other embodiments, the tumor antigen is a CD20 antigen. In other embodiments, the tumor antigen is a CD20 antigen and a CD79b antigen.

In other embodiments, the first antigen binding domain that binds CD3 epsilon and/or the second antigen binding domain that binds a tumor antigen comprises an scFv, (scFv) ₂ 、Fv、Fab、F(ab') ₂ Fd, dAb or VHH.

In other embodiments, the first antigen binding domain that binds CD3 epsilon and/or the second antigen binding domain that binds a tumor antigen comprises Fab.

In other embodiments, the first antigen binding domain that binds CD3 epsilon and/or the second antigen binding domain that binds tumor antigen comprises F (ab') ₂ 。

In other embodiments, the first antigen binding domain that binds CD3 epsilon and/or the second antigen binding domain that binds a tumor antigen comprises a VHH.

In other embodiments, the first antigen binding domain that binds CD3 epsilon and/or the second antigen binding domain that binds a tumor antigen comprises Fv.

In other embodiments, the first antigen binding domain that binds CD3 epsilon and/or the second antigen binding domain that binds a tumor antigen comprises Fd.

In other embodiments, the first antigen binding domain that binds CD3 epsilon and/or the second antigen binding domain that binds a tumor antigen comprises an scFv.

In other embodiments, the scFv comprises, from N-terminus to C-terminus, a VH, a first linker (L1) and a VL (VH-L1-VL) or comprises the VL, the L1 and the VH (VL-L1-VH).

In other embodiments, the L1 comprises about 5 to 50 amino acids.

In other embodiments, the L1 comprises about 5 to 40 amino acids.

In other embodiments, the L1 comprises about 10 to 30 amino acids.

In other embodiments, the L1 comprises about 10 to 20 amino acids.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 3.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 4.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 5.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 6.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 7.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 8.

In other embodiments, the L1 comprises the amino acid sequence of SEQ ID NO. 9.

In some embodiments, the first antigen binding domain that binds CD3 ε comprises HCDR1 of SEQ ID NO:70, HCDR2 of SEQ ID NO:71, HCDR3 of SEQ ID NO:72, 87, 90 or 86, LCDR1 of SEQ ID NO:79, LCDR2 of SEQ ID NO:80, and LCDR3 of SEQ ID NO: 81.

In other embodiments, the first antigen binding domain that binds CD3 ε comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 86, 79, 80 and 81, respectively.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as follows:

SEQ ID NOs 70, 71, 72, 79, 80 and 81, respectively;

70, 71, 87, 79, 80 and 81 respectively; or alternatively

SEQ ID NOS 70, 71, 90, 79, 80 and 81, respectively.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the VH of SEQ ID NO:55 and the VL of SEQ ID NO: 59.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the VH of SEQ ID NO:55 and the VL of SEQ ID NO: 58.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the VH of SEQ ID NO:54 and the VL of SEQ ID NO: 56.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the VH of SEQ ID NO:48 and the VL of SEQ ID NO: 58.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the VH of SEQ ID NO:88 and the VL of SEQ ID NO: 58.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the VH of SEQ ID NO:242 and the VL of SEQ ID NO: 58.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the VH of SEQ ID NO:55, 54 or 48, and the VL of SEQ ID NO:59, 58 or 56.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or 126.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO: 96.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 97.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 98.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO 99.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 100.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 101.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 102.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 103.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 104.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 105.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 106.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 107.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 108.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 109.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 110.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 112.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 113.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 114.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 115.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 116.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 117.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 118.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO: 119.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 120.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 121.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 122.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 123.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 124.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 125.

In other embodiments, the first antigen binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NO. 126.

In other embodiments, the second antigen binding domain that binds a tumor antigen is specific for PSMA.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1242 and LC of SEQ ID NO. 1243.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1244 and LC of SEQ ID NO. 1245.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1246 and LC of SEQ ID NO. 1247.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1248.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO: 1250.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1252.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1254.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1256.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1258.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO 1260.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1262.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO 1264.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1266.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1268.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1270.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO 1272.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO 1274.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO 1276.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO 1278.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1280.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1282.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1284.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1286.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1288.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1290.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1292.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1294.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO:1459 and LC of SEQ ID NO: 1460.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1461 and LC of SEQ ID NO. 1462.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO 1356 and LC of SEQ ID NO 1357.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO 1358 and LC of SEQ ID NO 1359.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1360 and LC of SEQ ID NO. 1361.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1362 and LC of SEQ ID NO. 1363.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1364 and LC of SEQ ID NO. 1365.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1366 and LC of SEQ ID NO. 1367.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1368 and LC of SEQ ID NO. 1369.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO 1370 and LC of SEQ ID NO 1371.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1372 and LC of SEQ ID NO. 1373.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO 1374 and LC of SEQ ID NO 1375.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO:1376 and LC of SEQ ID NO: 1377.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO 1378 and LC of SEQ ID NO 1379.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1380 and LC of SEQ ID NO. 1381.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1382 and LC of SEQ ID NO. 1383.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1384 and LC of SEQ ID NO. 1385.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1386 and LC of SEQ ID NO. 1387.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1388 and LC of SEQ ID NO. 1389.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1390 and LC of SEQ ID NO. 1391.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1392 and LC of SEQ ID NO. 1393.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO:1394 and LC of SEQ ID NO: 1395.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1396 and LC of SEQ ID NO. 1397.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1398 and LC of SEQ ID NO. 1399.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1400 and LC of SEQ ID NO. 1401.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1402 and LC of SEQ ID NO. 1403.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO:1404 and LC of SEQ ID NO: 1405.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO:1406 and LC of SEQ ID NO: 1407.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO:1408 and LC of SEQ ID NO: 1409.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1410 and LC of SEQ ID NO. 1411.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1412 and LC of SEQ ID NO. 1413.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO 1414 and LC of SEQ ID NO 1415.

In other embodiments, the second antigen binding domain that binds a tumor antigen is specific for CD79 b.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1489 and LC of SEQ ID NO. 1491.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1493 and LC of SEQ ID NO. 1495.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO. 1497 and LC of SEQ ID NO. 1499.

In other embodiments, the second antigen binding domain that binds a tumor antigen comprises HC of SEQ ID NO:1502 and LC of SEQ ID NO: 1499.

In other embodiments, the first antigen binding domain that binds CD3 epsilon is conjugated to a first immunoglobulin (Ig) constant region or fragment of a first Ig constant region and/or the second antigen binding domain that binds a tumor antigen is conjugated to a second immunoglobulin (Ig) constant region or fragment of a second Ig constant region.

In other embodiments, the fragment of the first Ig constant region and/or the fragment of the second Ig constant region comprises an Fc region.

In other embodiments, the fragment of the first Ig constant region and/or the fragment of the second Ig constant region comprises a CH2 domain.

In other embodiments, the fragment of the first Ig constant region and/or the fragment of the second Ig constant region comprises a CH3 domain.

In other embodiments, the fragment of the first Ig constant region and/or the fragment of the second Ig constant region comprises a CH2 domain and a CH3 domain.

In other embodiments, the fragment of the first Ig constant region and/or the fragment of the second Ig constant region comprises at least a portion of a hinge, a CH2 domain, and a CH3 domain.

In other embodiments, the multispecific protein further comprises a second linker (L2) between a first antigen-binding domain that binds CD3 epsilon and a first Ig constant region or fragment of a first Ig constant region and a second antigen-binding domain that binds a tumor antigen and a second Ig constant region or fragment of a second Ig constant region.

In other embodiments, the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region are IgG1, igG2, and IgG3 or IgG4 isotypes.

In other embodiments, the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region are IgG1 isotypes.

In other embodiments, the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region are IgG2 isotypes.

In other embodiments, the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region are IgG3 isotypes.

In other embodiments, the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region are of the IgG4 isotype.

The first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region can be further engineered as described herein.

In other embodiments, the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise at least one mutation that results in reduced binding of the multispecific protein to the fcγr.

In other embodiments, the at least one mutation that results in reduced binding of the multispecific protein to fcγr is selected from the group consisting of: F234A/L235A, L A/L235A, L A/L235A/D265S, V234A/G237A/P238S/H268A/V309L/A330S/P331S, F A/L235A, S P/F234A/L235A, N297A, V A/G237A, K T/E233P/L234V/L235A/G236-deletion/A327G/P331A/D365E/L358M, H268Q/V309L/A330S/P331S, S E/L328F, L F/L235E/D265A, L A/L235A/G237A/P238S/H268A/A330S/P331S, S P/F234A/L235A/G237A/P238S and S228P/F234A/L235A/G236-deletion/G237A/P238S, wherein residue numbering is performed according to the EU index.

In other embodiments, the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise at least one mutation that results in enhanced binding of the multispecific protein to an fcγ receptor (fcγr).

In other embodiments, the at least one mutation that results in enhanced binding of the multispecific protein to fcγr is selected from the group consisting of: S239D/I332E, S A/E333A/K334A, F L/R292P/Y300L, F243L/R292P/Y300L/P396L, F243L/R292P/Y300L/V305I/P396L and G236A/S239D/I332E, wherein residue numbering is according to the EU index.

In other embodiments, the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise at least one mutation that modulates the half-life of the multispecific protein.

In other embodiments, the at least one mutation that modulates the half-life of the multispecific protein is selected from the group consisting of: H435A, P257I/N434H, D376V/N434H, M Y/S254T/T256E/H433K/N434F, T308P/N434A and H435R, wherein residues are numbered according to the EU index.

In other embodiments, the multispecific protein comprises at least one mutation in the CH3 domain of the first Ig constant region or in the CH3 domain of a fragment of the first Ig constant region and/or comprises at least one mutation in the CH3 domain of the second Ig constant region or in the CH3 domain of a fragment of the second Ig constant region.

In other embodiments, the at least one mutation in the CH3 domain of the first Ig constant region or in the CH3 domain of the fragment of the first Ig constant region and/or the at least one mutation in the CH3 domain of the second Ig constant region or in the CH3 domain of the fragment of the second Ig constant region is selected from the group consisting of: T350V, L351Y, F A, Y407V, T366Y, T366W, T366L, T L, F405W, T394W, K392L, T394S, T394W, Y407T, Y A, T S/L368A/Y407V, L Y/F405A/Y407V, T I/K392M/T394W, F A/Y407V, T366L/K392M/T394W, T L/K392L/T394W, L57351Y/Y407A, L Y/Y407V, T A/K409F, T366V/K409F, T366A/K409F, T V/L351Y/F405A/Y407V and T350V/T366L/K392L/T394W, wherein residue numbering is according to EU index.

In other embodiments, the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutations:

l235a_l235a_d265s_t350v_l351 y_f455a_y407V in the first Ig constant region and l235a_l235s_t350v_t366 l_k404l_t394W in the second Ig constant region; or alternatively

L235a_l235a_d265s_t350v_t366l_k392l_t394W in the first Ig constant region and L235a_l235a_d265s_t350v_l351y_f405a_y407V in the second Ig constant region.

Trispecific antibodies

In some embodiments, provided herein are trispecific antibodies that bind to CD79b, CD20, and CD3, and trispecific binding fragments thereof. This can be accomplished, for example, by preparing a molecule comprising a first binding region that specifically binds CD79b, a second binding region that specifically binds CD3, and a third binding region that specifically binds CD20. These antigen binding regions may take any form that allows specific recognition of the target, e.g., the binding region may be or may include a heavy chain variable domain, fv (a combination of a heavy chain variable domain and a light chain variable domain), single chain Fv (scFv), fab, binding domains based on a type III fibronectin domain (such as from fibronectin or based on a consensus sequence of a type III domain from fibronectin, or from tenascin or based on a consensus sequence of a type III domain from tenascin, such as a Centyrin molecule from Janssen Biotech, inc., see, e.g., WO2010/051274 and WO 2010/093627). Thus, a trispecific molecule comprising three different antigen-binding regions that bind CD79b, CD20 and CD3, respectively, is provided.

In some embodiments, the cd79b×cd20×cd3 trispecific antibody comprises a first heavy chain (HC 1) and a Light Chain (LC) paired to form a first antigen binding site that specifically binds a first antigen, and a second heavy chain (HC 2) comprising a second antigen binding site that specifically binds a second antigen. HC1 or HC2 may further comprise a third antigen binding site that specifically binds a third antigen. HC1 and HC2 may each comprise a fragment crystallizable (Fc) domain comprising a CH2-CH3 domain. In a preferred embodiment, the CD79b x CD20 x CD3 multispecific antibody is a trispecific antibody comprising a CD79 b-specific arm comprising a first heavy chain (HC 1) and a Light Chain (LC) paired to form a first antigen-binding site that specifically binds CD79b, and a second heavy chain (HC 2) comprising a second antigen-binding site that specifically binds a second antigen, and HC1 or HC2 further comprises a third antigen-binding site that specifically binds a third antigen. In some embodiments, the second antigen is CD20 and the third antigen is CD3. In some embodiments, the second antigen is CD3 and the third antigen is CD20.

In some embodiments, HC2 comprises a third antigen binding site that specifically binds a third antigen. For example, HC2 can comprise, from N-terminus to C-terminus, a second antigen binding site, an Fc domain, a linker, and a third antigen binding site.

In some embodiments, HC1 comprises a third antigen binding site that specifically binds a third antigen. For example, HC1 can comprise, from N-terminus to C-terminus, a heavy chain variable domain (VH), CH1 domain, fc domain, linker, and a third antigen binding site associated with the first antigen binding site.

In one embodiment, the cd79b×cd20×cd3 multispecific antibody is a trispecific antibody comprising a CD79 b-specific arm comprising HC1 and LC and HC2, wherein HC1 and LC pair to form a first antigen binding site that specifically binds CD79b, HC2 comprises a second antigen binding site that specifically binds CD3, and HC2 further comprises a third antigen binding site that specifically binds CD 20.

In one embodiment, the cd79b×cd20×cd3 multispecific antibody is a trispecific antibody comprising a CD79 b-specific arm comprising HC1 and LC and HC2, wherein HC1 and LC pair to form a first antigen binding site that specifically binds CD79b, HC2 comprises a second antigen binding site that specifically binds CD20, and HC2 further comprises a third antigen binding site that specifically binds CD 3.

In one embodiment, the cd79b×cd20×cd3 multispecific antibody is a trispecific antibody comprising a CD79 b-specific arm comprising HC1 and LC and HC2, wherein HC1 and LC pair to form a first antigen binding site that specifically binds CD79b, HC2 comprises a second antigen binding site that specifically binds CD20, and HC1 further comprises a third antigen binding site that specifically binds CD 3.

In some embodiments, the first antigen binding site comprises an antigen binding fragment (Fab). In some embodiments, the second antigen binding site comprises a single chain variable fragment (scFv). In some embodiments, the third antigen binding site comprises a single chain variable fragment (scFv).

In one embodiment, the CD79b binding arm comprises an antigen binding fragment (Fab), the CD3 binding arm comprises a single chain variable fragment (scFv), and the CD20 binding arm also comprises a single chain variable fragment (scFv).

Exemplary heavy and light chains of exemplary trispecific binding proteins of the disclosure are shown in table 31.

Generation of multispecific proteins comprising antigen-binding fragments that bind CD3 epsilon。

The antigen binding fragments of the present disclosure that bind CD3 epsilon may be engineered into multispecific antibodies, which are also encompassed within the scope of the present invention.

The antigen binding fragment that binds CD3 epsilon can be engineered into a full length multispecific antibody generated using Fab arm exchange, wherein substitutions are introduced into the two monospecific bivalent antibodies within the Ig constant region CH3 domain that facilitate Fab arm exchange in vitro. In the method, two monospecific bivalent antibodies are engineered with certain substitutions at the CH3 domain that promote heterodimer stability; incubating the antibodies under reducing conditions sufficient to disulfide isomerize cysteines in the hinge region; thereby generating bispecific antibodies by Fab arm exchange. The incubation conditions are most desirably restorable to non-reducing conditions. Exemplary reducing agents that may be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris (2-carboxyethyl) phosphine (TCEP), L-cysteine and beta-mercaptoethanol, preferably reducing agents selected from the group consisting of 2-mercaptoethylamine, dithiothreitol and tris (2-carboxyethyl) phosphine. For example, the following conditions may be used: incubation is carried out for at least 90 minutes in the presence of at least 25mM 2-MEA or in the presence of at least 0.5mM dithiothreitol at a pH of 5-8, e.g.pH 7.0 or pH7.4, at a temperature of at least 20 ℃.

CH3 mutations that can be used include, for example, knob hole mutations (Genntech), electrostatic matching mutations (Chugai, amgen, novoNordisk, oncomeded), strand exchange engineering domain bodies (SE)EDbody)(EMD Serono)、Mutation (Genmab) and other asymmetric mutation (e.g., zymeworks).

Knob hole mutations are disclosed in, for example, WO1996/027011 and include mutations at the interface of CH3 regions, wherein amino acids with small side chains (holes) are introduced into a first CH3 region and amino acids with large side chains (knobs) are introduced into a second CH3 region, resulting in preferential interactions between the first CH3 region and the second CH3 region. Exemplary CH3 region mutations that form knobs and holes are T366Y/F405A, T W/F405W, F W/Y407A, T W/Y407T, T394S/Y407A, T366W/T394S, F W/T394S and T366W/T366S_L368A_Y407V.

The formation of heavy chain heterodimers can be facilitated by substitution of positively charged residues on the first CH3 region and negatively charged residues on the second CH3 region using electrostatic interactions, as described in US2010/0015133, US2009/0182127, US2010/028637 or US 2011/0123032.

Other asymmetric mutations that can be used to promote heavy chain heterodimerization are L351Y_F405A_Y407V/T394W, T I_K392M_T394W/F405A_Y407V, T366L_K392M_T394W/F405A_Y407V, L351Y 407A/T366A_K409F, L351Y 407A/T366V_K409F, Y407 A_K409F or T350V_L351Y_F405A_Y 407V/T366 L_K392L_T394W as described in US2012/0149876 or US2013/0195849 (zymews).

The SEEDbody mutation involves substitution of selected IgG residues with IgA residues to promote heavy chain heterodimerization as described in US 20070287170.

Other exemplary mutations that may be used are R409D_K370E/D399K_E357K, S C_T366W/Y349C_T366S_L368A_Y407V, Y349C_T366W/S354C_T366S_L368A_Y407V, T366K/L351D, L K/Y349E, L K/Y349D, L K/L368E, L Y_Y407A/T366A_K409F, L Y351Y 407A/T366V_K409F, K392D/D3995392D/E356K, K E_D282K 322D/D239K_E240K 292D, K D_K409D 356K_D399K as described in WO2007/147901, WO 2011/143545, WO2013157954, WO 628832 and US 2018/01149K.

Mutations (Genmab) are disclosed in, for example, US9150663 and US2014/0303356, and include the mutations F405L/K409R, wild-type/F405 L_R409K, T350I_K370T_F405L/K409R, K W/K409R, D399AFGHILMNRSTVWY/K409R, T366ADEFGHILMQVY/K409R, L368ADEGHNRSTVQ/K409AGRH, D399FHKRQ/K409AGRH, F405IKLSTVW/K409AGRH and Y407LWQ/K409AGRH.

Additional bispecific or multispecific structures into which antigen binding domains that bind CD3 epsilon can be incorporated include dual variable domain immunoglobulins (DVD) (international patent publication No. WO2009/134776; DVD is a full length antibody comprising a heavy chain with a VH 1-linker-VH 2-CH structure and a light chain with a VL 1-linker-VL 2-CL structure; linker is optional), structures comprising multiple dimerization domains to connect two antibody arms with different specificities such as leucine zipper or collagen dimerization domains (international patent publication No. WO2012/022811, U.S. patent No. 5,932,448 and U.S. patent No. 6,833,441), two or more domain antibodies (dAb) conjugated together, diabodies, heavy chain-only antibodies such as camelidae antibodies and engineered camelidae antibodies, dual Targeting (DT) -antibodies (GSK/domans), diabodies (Genentech), cross-links Mab (Karmanos Cancer Center), 2 (F-Star) and CovX-bodies (cox/pfzer), diabodies (IgG/35 n), diabodies (sciv) and sciv-92 (sciv) and (sciv) diabodies (sciv) 96-35 n), diabodies (sciv) and (sciv-35 Fc) and (sciv-96-35 b) fusion technologies Bifunctional or Bis-Fab (Genentech), dock-lock (DNL) (immunoMedics), bivalent bispecific (Biotech) and Fab-Fv (UCB-Celltech). ScFv-based, diabody-based domain antibodies include, but are not limited to, bispecific T cell engagers (BiTE) (Micromet), tandem diabodies (Tandab) (affied), amphipathic retargeting techniques (DART) (macrographics), single chain diabodies (Academic), TCR-like antibodies (AIT, receptorLogics), human serum albumin ScFv fusions (Merrimack) and COMBODY (Epigen Biotech), double targeting nanobodies (Ablynx), double targeting heavy chain domain-only antibodies.

The antigen binding domains of the present disclosure that bind CD3 epsilon can also be engineered into a multispecific protein that comprises three polypeptide chains. In such designs, at least one antigen binding domain is in the form of an scFv. Exemplary designs include (where "1" indicates a first antigen binding domain, "2" indicates a second antigen binding domain, and "3" indicates a third antigen binding domain:

design 1: a chain) scFv1-CH2-CH3; chain B) VL2-CL; c chain) VH2-CH 1-hinge-CH 2-CH3

Design 2: a chain) scFv 1-hinge-CH 2-CH3; chain B) VL2-CL; c chain) VH2-CH 1-hinge-CH 2-CH3

Design 3: a chain) scFv1-CH 1-hinge-CH 2-CH3; chain B) VL2-CL; c chain) VH2-CH 1-hinge-CH 2-CH3

Design 4: a chain) CH2-CH3-scFv1; chain B) VL2-CL; c chain) VH2-CH 1-hinge-CH 2-CH3

CH3 engineering can be incorporated into designs 1-4, such as the mutation L351Y_F405A_Y407V/T394W, T I_K392M_T394W/F405A_Y407V, T366L_K392M_T394W/F405A_Y407V, L351Y 407A/T366A_K409F, L351Y 407A/T366V_K409F, Y407 A_K409F or T350V_L351Y_F405A_Y407 V_T366L_K392L_T394W, as described in US2012/0149876 or US2013/0195849 (Zymeworks).

Isotype, allotype and Fc engineering

The Ig constant region or fragment of an Ig constant region, such as the Fc region present in the proteins of the present disclosure, may be of any isotype or isotype.

In other embodiments, the Ig constant region or fragment of an Ig constant region is an IgG1 isotype.

In other embodiments, the Ig constant region or fragment of an Ig constant region is an IgG2 isotype.

In other embodiments, the Ig constant region or fragment of an Ig constant region is an IgG3 isotype.

In other embodiments, the Ig constant region or fragment of an Ig constant region is an IgG4 isotype.

The Ig constant region or fragment of an Ig constant region can be any allotype. The allotype is expected to have no effect on properties of the Ig constant region such as binding or Fc-mediated effector function. Immunogenicity of therapeutic proteins comprising Ig constant regions or fragments thereof is associated with increased infusion reaction risk and reduced duration of therapeutic response (Baert et al, (2003) N Engl J Med 348:602-08). The extent to which a therapeutic protein comprising an Ig constant region or fragment thereof induces an immune response in a host may be determined in part by the allotype of the Ig constant region (Stickler et al, (2011) Genes and Immunity 12:213-21). Ig constant region allotypes are related to amino acid sequence variations at specific positions in the constant region sequence of an antibody. Table 3 shows selected IgG1, igG2 and IgG4 allotypes.

Table 3.

C-terminal lysines (CTLs) can be removed from Ig constant regions by endogenous circulating carboxypeptidase in the blood stream (Cai et al, (2011) Biotechnol Bioeng 108:404-412). During the preparation, the extracellular Zn can be controlled ²⁺ EDTA or EDTA-Fe ³⁺ Is controlled to less than a maximum level as described in U.S. patent publication US 20140273092. The CTL content in the protein can be determined using known methods.

In other embodiments, the antigen binding fragment that binds CD3 epsilon conjugated to an Ig constant region has a C-terminal lysine content of about 10% to about 90%. In other embodiments, the C-terminal lysine content is about 20% to about 80%. In other embodiments, the C-terminal lysine content is about 40% to about 70%. In other embodiments, the C-terminal lysine content is about 55% to about 70%. In other embodiments, the C-terminal lysine content is about 60%.

The antigen binding domain that binds CD3 epsilon conjugated to an Ig constant region or fragment of an Ig constant region can be subjected to Fc region mutations to modulate its effector functions, such as ADCC, ADCP and/or pharmacokinetic properties. This can be achieved by introducing a mutation into the Fc that modulates binding of the mutated Fc to activated fcγr (fcγri, fcγriia, fcγriii), inhibitory fcγriib and/or FcRn.

In other embodiments, the CD3 epsilon binding antigen binding domain conjugated to an Ig constant region or fragment of an Ig constant region comprises at least one mutation in the Ig constant region or fragment of an Ig constant region.

In other embodiments, the at least one mutation is in the Fc region.

In other embodiments, the antigen binding domain that binds CD3 epsilon conjugated to an Ig constant region or fragment of an Ig constant region comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen mutations in the Fc region.

In other embodiments, the CD3 epsilon binding antigen binding domain conjugated to an Ig constant region or fragment of an Ig constant region comprises at least one mutation in the Fc region that modulates the binding of an antibody to FcRn.

Fc positions that can be mutated to modulate half-life (e.g., binding to FcRn) include positions 250, 252, 253, 254, 256, 257, 307, 376, 380, 428, 434, and 435. Exemplary mutations that may be generated, alone or in combination, are the mutations T250Q, M Y, I253A, S T, T E, P257I, T307A, D35307V, E380A, M428L, H433K, N434S, N434A, N H, N434F, H435A and H435R. Exemplary single mutations or combination mutations to increase half-life can be generated as mutations M428L/N434S, M Y/S254T/T256E, T Q/M428L, N434A and T307A/E380A/N434A. Exemplary single mutations or combined mutations to reduce half-life can be generated as mutations H435A, P I/N434H, D376V/N434H, M Y/S254T/T256E/H433K/N434F, T308P/N434A and H435R.

In other embodiments, the antigen binding domain that binds CD3 epsilon conjugated to an Ig constant region or fragment of an Ig constant region comprises an M252Y/S254T/T256E mutation.

In other embodiments, the CD3 epsilon binding antigen binding domain conjugated to an Ig constant region or fragment of an Ig constant region comprises at least one mutation in the Fc region that reduces binding of the protein to an activated fcγ receptor (fcγr) and/or reduces Fc effector functions such as C1q binding, complement Dependent Cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), or phagocytosis (ADCP).

Fc positions that can be mutated to reduce binding of a protein to an activated fcγr and subsequently reduce effector function include positions 214, 233, 234, 235, 236, 237, 238, 265, 267, 268, 270, 295, 297, 309, 327, 328, 329, 330, 331 and 365. Exemplary mutations that may be generated individually or in combination are the mutations K214T, E233P, L234V, L234 3834 deletion, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S in IgG1, igG2, igG3 or IgG4, the mutations K214T, E233P, L A, Q, 32309 327S, L F, A S and P331S. An exemplary combinatorial graph of proteins with reduced ADCC was generated for mutation L234A/L235A, igG1 at IgG1, mutation V234A/G237A/P238S/H330A/V309L/V309A/V330S/P331S, igG at C.sub.1, mutation S228P/F234A/L235A/235A at C.sub.58 4 at C.sub.35, mutation K214T/E233P/L234V/L234A/G236 at C.sub.97 69 1 at C.sub.234A/G237A, igG1 at C.sub.234A/L234A/V309L/A330S/P331A/L35 C.sub.sub.1 at C.sub.8A/P331A/L35S/C.sub.sub.sub.1 at C.sub.234A/L234A/L235A/G237A/P.sub.sub.35 C.sub.35S 35 C.sub.sub.1 at C.sub.234A/L235A/G235A/P.sub.sub.sub.35S 48A/F.sub.35S 48A/F.sub.sub.sub.35 S.35 C.sub.35S 48A/F.sub.2348A/F.sub.2335 S.234 at C.sub.L 235A/F.2335A/L235A/L35.sub.sub.L 35.L 35.sub.L 35.L 35.35.L 35.L 35.35.35.L.35.35.35.35.35 2. Hybrid IgG2/4Fc domains, such as Fc with residues 117-260 from IgG2 and residues 261-447 from IgG4, may also be used.

An exemplary mutation that produces a protein with reduced CDC is the K322A mutation.

Well known S228P mutations can be made in IgG4 antibodies to enhance IgG4 stability.

In other embodiments, the antigen binding domain that binds CD3 epsilon conjugated to an Ig constant region or fragment of an Ig constant region comprises at least one mutation selected from the group consisting of: K214T, E233P, L234V, L A, G4 missing, V234A, F234A, L235A, G237A, P A, P238S, D265 267E, H268A, H268Q, Q268 297A, A Q, P329A, Q295A, V309L, A S, L F, K322, a330S and P331S.

In other embodiments, the antigen binding domain that binds CD3 epsilon conjugated to an Ig constant region or fragment of an Ig constant region comprises an L234A/L235A/D265S mutation.

In other embodiments, the antigen binding domain of binding CD3 epsilon conjugated to an Ig constant region or fragment of an Ig constant region comprises an L234A/L235A mutation.

In other embodiments, the CD3 epsilon binding antigen binding domain conjugated to an Ig constant region or fragment of an Ig constant region comprises at least one mutation in the Fc region that enhances binding of the protein to an fcγ receptor (fcγr) and/or enhances Fc effector functions such as C1q binding, complement Dependent Cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), and/or phagocytosis (ADCP).

Fc positions that can be mutated to increase binding of a protein to an activated fcγr and/or enhance Fc effector function include positions 236, 239, 243, 256, 290, 292, 298, 300, 305, 312, 326, 330, 332, 333, 334, 345, 360, 339, 378, 396, or 430 (residue numbering according to EU index). Exemplary mutations that may be made, alone or in combination, are G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y L, V305L, K A, A K, I332E, E333A, K334A, A339T and P396L. Exemplary combinations of proteins that produce increased ADCC or ADCP are mutated to S239D/I332E, S A/E333A/K334A, F243L/R292P/Y300L, F243L/R292P/Y300L/P396L, F243L/R292P/Y300L/V305I/P396L and G236A/S239D/I332E.

Fc positions that may be mutated to enhance CDC include positions 267, 268, 324, 326, 333, 345, and 430. Exemplary mutations that may be generated, either alone or in combination, are S267E, F1268F, S T, K A, K326W, E A, E345K, E Q, E345R, E Y, E S, E430F and E430T. An exemplary combination mutation that produces a protein with increased CDC is K326A/E333A, K W/E333A, H268F/S324T, S E/H268F, S E/S324T and S267E/H268F/S324T.

Specific mutations described herein are mutations compared to the IgG1, igG2, and IgG4 wild-type amino acid sequences of SEQ ID NOs 237, 238, and 239, respectively.

SEQ ID NO. 237, wild type IgG1

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

238, wild type IgG2

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

239 of SEQ ID NO; wild type IgG4

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Binding of antibodies to fcγr or FcRn can be assessed using flow cytometry on cells engineered to express each receptor. In an exemplary binding assay, 2×10 will be ⁵ Individual cells/wells were seeded into 96-well plates and blocked in BSA staining buffer (BD Biosciences, san Jose, USA) for 30min at 4 ℃. Cells were incubated with test antibodies for 1.5 hours at 4℃on ice. After washing twice with BSA staining buffer, the cells were incubated with R-PE labeled anti-human IgG secondary antibody (Jackson Immunoresearch Laboratories) for 45min at 4 ℃.Cells were washed twice in staining buffer and then resuspended in 150 μl of staining buffer (Cell Signaling Technology, danvers, USA) containing 1:200 diluted DRAQ7 live/dead stain. PE and DRAQ7 signals of stained cells were detected by Miltenyi MACSQuant flow cytometry (Miltenyi Biotec, auburn, USA) using the B2 and B4 channels, respectively. Viable cells were gated according to the DRAQ7 exclusion method and the geometric mean fluorescence signal of at least 10,000 viable events collected was determined. Analysis was performed using FlowJo software (Tree Star). Data are plotted as log antibody concentration versus mean fluorescence signal. Nonlinear regression analysis was performed.

Sugar engineering

The ability of the antigen binding domain conjugated to an Ig constant region or fragment of an Ig constant region to bind CD3 epsilon to mediate ADCC can be enhanced by engineering the oligosaccharide component of the Ig constant region or fragment of the Ig constant region. Human IgG1 or IgG3 is well known for the N-glycosylation of most glycans in the form of the double branch G0, G0F, G1, G1F, G2 or G2F at Asn 297. The non-engineered CHO cells can produce a protein comprising an Ig constant region, typically having a glycan fucose content of about at least 85%. Removal of core fucose from a dual antenna complex oligosaccharide attached to a CD3 epsilon binding antigen binding domain conjugated to an Ig constant region or fragment of an Ig constant region enhances ADCC of a protein via improved fcyriiia binding without altering antigen binding or CDC activity. Such proteins can be achieved using different methods that have been reported to cause successful expression of relatively high defucosylated immunoglobulins with double antenna complex Fc oligosaccharides, such as controlling culture osmotic pressure (Konno et al Cytotechnology 64 (: 249-65, 2012), applying variant CHO line Lec13 as a host cell line (thields et al, J Biol Chem 277:26733-26740,2002), applying variant CHO line EB66 as a host cell line (Olivier et al, MAbs;2 (4): 405-415,2010; PMID: 20562582), the use of the rat hybridoma cell line YB2/0 as a host cell line (Shinkawa et al, J Biol Chem 278:3466-3473,2003), the introduction of small interfering RNAs specific for the 1, 6-fucosyltransferase (FUT 8) gene (Mori et al, biotechnol Bioeng:901-908,2004), or the co-expression of beta-1, 4-N-acetylglucosaminyltransferase III and Golgi alpha-mannosidase II or the potent alpha-mannosidase I inhibitor several fubase (Ferrara et al, J Biol Chem 281:5032-5036,2006, ferrara et al, biotechnol Bioeng:851-861, 2006; xhou et al, biotechnol Bioeng:652-65,2008).

In other embodiments, the CD3 epsilon binding antigen binding domain conjugated to an Ig constant region or fragment of an Ig constant region of the present disclosure has a biantennary glycan structure with a fucose content of about 1% to about 15%, e.g., about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In other embodiments, the antigen binding domain that binds CD3 epsilon conjugated to an Ig constant region or fragment of an Ig constant region has a glycan structure with a fucose content of about 50%, 40%, 45%, 40%, 35%, 30%, 25%, or 20%.

"fucose content" means the amount of fucose monosaccharides within the sugar chain at Asn 297. The relative amount of fucose is the percentage of fucose-containing structures relative to all sugar structures. These can be characterized and quantified by a variety of methods, such as: 1) MALDI-TOF using samples treated with N-glycosidase F (e.g., complex, mixed and oligomannose and high mannose structures), as described in International patent publication No. WO2008/077546 2; 2) Detection/quantification by enzymatic release of Asn297 glycans followed by derivatization and detection by HPLC with fluorescence detection (UPLC) and/or HPLC-MS (UPLC-MS); 3) Intact protein analysis was performed on native or reduced mabs with or without Endo S or other enzymes that cleave between the first GlcNAc monosaccharide and the second GlcNAc monosaccharide, leaving fucose attached to the first GlcNAc; 4) Digestion of the mAb into constituent peptides by enzymatic digestion (e.g., trypsin or endopeptidase Lys-C), followed by separation, detection and quantification by HPLC-MS (UPLC-MS); 5) mAb oligosaccharides were separated from mAb proteins by specific enzymatic deglycosylation with PNGase F at Asn 297. The oligosaccharides thus released can be fluorophore-labeled, isolated and identified by a variety of complementary techniques that allow for: fine characterization of the glycan structure by comparing experimental mass with theoretical mass using Matrix Assisted Laser Desorption Ionization (MALDI) mass spectrometry; determining the sialylation degree by ion exchange HPLC (GlycoSep C); isolating and quantifying the oligosaccharide form according to the hydrophilicity standard by normal phase HPLC (GlycoSep N); and separating and quantifying oligosaccharides by high-efficiency capillary electrophoresis laser induced fluorescence (HPCE-LIF).

As used herein, "low fucose" or "low fucose content" means that the antigen binding domain that binds CD3 epsilon conjugated to an Ig constant region or fragment of an Ig constant region has a fucose content of about 1% to 15%.

As used herein, "normal fucose" or "normal fucose content" refers to an antigen binding domain that binds CD3 epsilon conjugated to an Ig constant region or fragment of an Ig constant region having a fucose content of about 50% or more, typically about 80% or more, or 85% or more.

Anti-idiotype antibody

An anti-idiotype antibody is an antibody that specifically binds to the antigen binding domain of the present disclosure that binds CD3 epsilon.

The invention also provides an anti-idiotype antibody that specifically binds to the antigen binding domain of the present disclosure that binds CD3 epsilon.

The invention also provides an anti-idiotype antibody that specifically binds to an antigen binding domain that binds CD3 epsilon comprising:

VH of SEQ ID NO. 55 and VL of SEQ ID NO. 59;

VH of SEQ ID NO. 55 and VL of SEQ ID NO. 58;

VH of SEQ ID NO. 54 and VL of SEQ ID NO. 56;

VH of SEQ ID NO. 48 and VL of SEQ ID NO. 58;

the VH of SEQ ID NO. 88 and the VL of SEQ ID NO. 58; or alternatively

The VH of SEQ ID NO. 242 and the VL of SEQ ID NO. 58.

An anti-idiotype (Id) antibody is an antibody that recognizes an epitope (e.g., a paratope or CDR) of the antibody. The Id antibody may be antigen blocked or unblocked. The antigen block Id can be used to detect a free antigen binding domain (e.g., an antigen binding domain of the present disclosure that binds CD3 epsilon) in a sample. The non-blocking Id may be used to detect total antibodies (free, partially bound to antigen, or fully bound to antigen) in the sample. Id antibodies can be prepared by immunizing an animal from which the anti-Id is being prepared with an antibody.

The anti-Id antibody may also be used as an immunogen to induce an immune response in another animal, thereby producing a so-called anti-Id antibody. The anti-Id may have an epitope that is identical to the original antigen binding domain that induced the anti-Id. Thus, by using antibodies directed against the idiotype determinants of the antigen binding domain, other clones expressing the same specific antigen binding domain can be identified. The anti-Id antibody may be altered (thereby producing an anti-Id antibody variant) and/or derivatized by any suitable technique, such as those described elsewhere herein.

Immunoconjugates

The antigen binding domain that binds CD3 epsilon, a protein comprising an antigen binding domain that binds CD3 epsilon, or a multispecific protein comprising an antigen binding domain that binds CD3 epsilon (collectively referred to herein as CD3 epsilon binding proteins) of the present disclosure may be conjugated to a heterologous molecule.

In other embodiments, the heterologous molecule is a detectable label or a cytotoxic agent.

The invention also provides an antigen binding domain that binds CD3 epsilon conjugated to a detectable label.

The invention also provides a protein comprising an antigen binding domain conjugated to a detectable label that binds CD3 epsilon.

The invention also provides a multispecific protein comprising an antigen-binding domain conjugated to a detectable label that binds CD3 epsilon.

The invention also provides an antigen binding domain that binds CD3 epsilon conjugated to a cytotoxic agent.

The invention also provides a protein comprising an antigen binding domain that binds CD3 epsilon conjugated to a cytotoxic agent.

The invention also provides a multispecific protein comprising an antigen-binding domain that binds CD3 epsilon conjugated to a cytotoxic agent.

The CD3 epsilon binding proteins of the present disclosure can be used to direct therapeutic agents to cells expressing tumor antigens. Alternatively, cells expressing CD3 epsilon can be targeted with the CD3 epsilon binding proteins of the present disclosure, coupled with a therapeutic agent that is intended to modify cellular function once internalized.

In other embodiments, the detectable label is also a cytotoxic agent.

The CD3 epsilon binding proteins of the present disclosure conjugated to a detectable label can be used to evaluate the expression of CD3 epsilon on a variety of samples.

Detectable labels include compositions that when conjugated to the CD3 epsilon binding proteins of the present disclosure, render the CD3 epsilon binding proteins of the present disclosure detectable via spectroscopic, photochemical, biochemical, immunochemical, or chemical means.

Exemplary detectable labels include radioisotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in ELISA), biotin, digoxin, haptens, luminescent molecules, chemiluminescent molecules, fluorescent dyes, fluorophores, fluorescence quenchers, colored molecules, radioisotopes, scintillators, avidin, streptavidin, protein a, protein G, antibodies or fragments thereof, polyhistidine, ni ²⁺ Flag tag, myc tag, heavy metals, enzymes, alkaline phosphatase, peroxidase, luciferase, electron donor/acceptor, acridinium ester and colorimetric substrate.

The detectable label may spontaneously emit a signal, for example when the detectable label is a radioisotope. In other cases, the detectable label signals as a result of being stimulated by an external field.

Exemplary radioisotopes may be gamma emitting, auger emitting, beta emitting, alpha emitting, or positron emitting radioisotopes. Exemplary radioisotopes include ³ H、 ¹¹ C、 ¹³ C、 ¹⁵ N、 ¹⁸ F、 ¹⁹ F、 ⁵⁵ Co、 ⁵⁷ Co、 ⁶⁰ Co、 ⁶¹ Cu、 ⁶² Cu、 ⁶⁴ Cu、 ⁶⁷ Cu、 ⁶⁸ Ga、 ⁷² As、 ⁷⁵ Br、 ⁸⁶ Y、 ⁸⁹ Zr、 ⁹⁰ Sr、 ^94m Tc、 ^99m Tc、 ¹¹⁵ In、 ¹²³ 1、 ¹²⁴ 1、 ¹²⁵ I、 ¹³¹ 1、 ²¹¹ At、 ²¹² Bi、 ²¹³ Bi、 ²²³ Ra、 ²²⁶ Ra、 ²²⁵ Ac and ²²⁷ Ac。

exemplary metal atoms are metals having an atomic number greater than 20, such as a calcium atom, scandium atom, titanium atom, vanadium atom, chromium atom, manganese atom, iron atom, cobalt atom, nickel atom, copper atom, zinc atom, gallium atom, germanium atom, arsenic atom, selenium atom, bromine atom, krypton atom, rubidium atom, strontium atom, yttrium atom, zirconium atom, niobium atom, molybdenum atom, technetium atom, ruthenium atom, rhodium atom, palladium atom, silver atom, cadmium atom, indium atom, tin atom, antimony atom, tellurium atom, iodine atom, xenon atom, cesium atom, barium atom, lanthanum atom, hafnium atom, tantalum atom, tungsten atom, rhenium atom, osmium atom, iridium atom, platinum atom, gold atom, mercury atom, thallium atom, lead atom, bismuth atom, francium atom, radium atom, actinium atom, cerium atom, praseodymium atom, neodymium atom, samarium atom, europium atom, gadolinium atom, terbium atom, holmium atom, thulium atom, ytterbium atom, actinium atom, thorium atom, promiscum atom, neptunium atom, neptunum atom, americium atom, uranium atom, or calium atom.

In other embodiments, the metal atom may be an alkaline earth metal having an atomic number greater than twenty.

In other embodiments, the metal atom may be a lanthanide.

In other embodiments, the metal atom may be an actinide.

In other embodiments, the metal atom may be a transition metal.

In other embodiments, the metal atom may be lean.

In other embodiments, the metal atoms may be gold atoms, bismuth atoms, tantalum atoms, and gadolinium atoms.

In other embodiments, the metal atom may be a metal having an atomic number of 53 (i.e., iodine) to 83 (i.e., bismuth).

In other embodiments, the metal atoms may be atoms suitable for magnetic resonance imaging.

The metal atom may be a metal ion in the +1, +2 or +3 oxidation state, such as Ba ²⁺ 、Bi ³⁺ 、Cs ⁺ 、Ca ²⁺ 、Cr ²⁺ 、Cr ³⁺ 、Cr ⁶⁺ 、Co ²⁺ 、Co ³⁺ 、Cu ⁺ 、Cu ²⁺ 、Cu ³⁺ 、Ga ³⁺ 、Gd ³⁺ 、Au ⁺ 、Au ³⁺ 、Fe ²⁺ 、Fe ³⁺ 、F ³⁺ 、Pb ²⁺ 、Mn ²⁺ 、Mn ³⁺ 、Mn ⁴⁺ 、Mn ⁷⁺ 、Hg ²⁺ 、Ni ²⁺ 、Ni ³⁺ 、Ag ⁺ 、Sr ²⁺ 、Sn ²⁺ 、Sn ⁴⁺ And Zn ²⁺ . The metal atoms may include metal oxides such as iron oxide, manganese oxide, or gadolinium oxide.

Suitable dyes include any commercially available dye such as 5 (6) -carboxyfluorescein, IRDye680RD maleimide or IRDye 800CW, ruthenium polypyridine dye, and the like.

Suitable fluorophores are Fluorescein Isothiocyanate (FITC), thiosemicarbazide fluorescein, rhodamine, texas red, cyDye (e.g., cy3, cy5, cy 5.5), alexa Fluor (e.g., alexa488, alexa555, alexa594; alexa 647), near Infrared (NIR) (700 nm to 900 nm) fluorescent dyes, as well as carbocyanine and aminostyryl dyes.

The antigen binding domain conjugated to a detectable label that binds CD3 epsilon may be used as an imaging agent.

Proteins comprising an antigen binding domain conjugated to a detectably labeled binding CD3 epsilon may be used as imaging agents.

Multispecific proteins comprising an antigen-binding domain conjugated to a detectably labeled binding CD3 epsilon may be used as imaging agents.

In other embodiments, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope (i.e., a radio conjugate).

In other embodiments, the cytotoxic agent is daunorubicin, doxorubicin, methotrexate, vindesine, a bacterial toxin (such as diphtheria toxin), ricin, geldanamycin, maytansinoid, or calicheamicin. Cytotoxic agents may elicit their cytotoxic and cytostatic effects through mechanisms including tubulin binding, DNA binding or topoisomerase inhibition.

In other embodiments, the cytotoxic agent is an enzymatically active toxin, such as diphtheria chain, a non-binding active fragment of diphtheria toxin, an exotoxin a chain (from pseudomonas aeruginosa (Pseudomonas aeruginosa)), a ricin a chain, abrin a chain, a pristina chain, α -sarcina, a tung (Aleurites fordii) protein, a caryophyllin protein, a pokeweed (Phytolaca americana) protein (PAPI, PAPII, and PAP-S), a balsam pear (momordica charantia) inhibitor, jatrophin, crootoxin, a verruca (sapaonaria officinalis) inhibitor, gelonin, mitoxin, restrictocin, phenomycin, enomycin, and trichothecene family compounds.

In other embodiments, the cytotoxic agent is a radionuclide, such as ²¹² Bi、 ¹³¹ I、 ¹³¹ In、 ⁹⁰ Y and ¹⁸⁶ Re。

in other embodiments, the cytotoxic agent is dolastatin or dolastatin peptide analogs and derivatives, auristatin or monomethyl auristatin phenylalanine. Exemplary molecules are disclosed in U.S. Pat. nos. 5,635,483 and 5,780,588. Dolastatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al, (2001) Antimicrob Agents and chemother.45 (12): 3580-3584), and have anti-cancer and anti-fungal activity. The dolastatin or auristatin drug moiety can be attached to the antibodies of the invention through the N (amino) or C (carboxyl) terminus of the peptide drug moiety (WO 02/088172) or via any cysteine engineered into the antibody.

The CD3 epsilon binding proteins of the present disclosure can be conjugated to a detectable label using known methods.

In other embodiments, the detectable label is complexed with a chelator.

In other embodiments, the detectable label is conjugated to the CD3 epsilon binding protein of the present disclosure via a linker.

The detectable label or cytotoxic moiety may be attached directly or indirectly to the CD3 epsilon binding protein of the present disclosure using known methods. Suitable linkers are known in the art and include, for example, prosthetic groups, derivatives of non-phenolic linkers (N-succinimidyl-benzoate; dodecaborate), chelating moieties of both macrocyclic chelators and acyclic chelators, derivatives such as 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA), derivatives of diethylenetriamine pentaacetic acid (DTPA), derivatives of S-2- (4-isothiocyanatobenzyl) -1,4, 7-triazacyclononane-1, 4, 7-triacetic acid (NOTA) and derivatives of 1,4,8, 11-tetraazacyclododecane-1, 4,8, 11-tetraacetic acid (TETA), derivatives of N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiophene (IT), bifunctional derivatives such as hexamethylenediimine HCl, active esters such as bissuccinimidyl suberate), aldehydes such as glutaraldehyde, bis (bis-azido) and bis (dinitrophenyl) bis (2, 4,8, 11-tetraazacyclododecane-1, 4-tetraacetic acid (TETA), bis (bis-dinitrophenyl) and bis (2, 4,8, 11-tetraacetic acid) such as bis (2-pyridyldithiol) propionate, bis (4, bis-succinimido) and other bis (2-fluoro) derivatives. Suitable peptide linkers are well known.

In other embodiments, the CD3 epsilon binding protein of the present disclosure is removed from the blood via renal clearance.

Kit for detecting a substance in a sample

The invention also provides a kit comprising an antigen binding domain that binds CD3 epsilon.

The invention also provides a kit comprising a protein comprising an antigen binding domain that binds CD3 epsilon.

The invention also provides a kit comprising a multispecific protein comprising an antigen-binding domain that binds CD3 epsilon.

The kit may be used for therapeutic purposes and as a diagnostic kit.

The kit can be used to detect the presence of CD3 epsilon in a sample.

In other embodiments, the kit comprises a CD3 epsilon binding protein of the present disclosure and reagents for detecting the CD3 epsilon binding protein. The kit may comprise one or more other elements, including: instructions for use; other agents, such as labels, therapeutic agents, or agents useful for chelating or otherwise coupling antibodies to labels or therapeutic agents, or radioprotective compositions; a device or other material for preparing to administer the antibody; a pharmaceutically acceptable carrier; and devices or other materials for administration to a subject.

In other embodiments, the kit comprises in a container an antigen binding domain that binds CD3 epsilon and instructions for use of the kit.

In other embodiments, the kit comprises in a container a protein comprising an antigen binding domain that binds CD3 epsilon and instructions for use of the kit.

In other embodiments, the kit comprises in a container a multispecific protein comprising an antigen-binding domain that binds CD3 epsilon and instructions for use of the kit.

In other embodiments, the antigen binding domain that binds CD3 epsilon in the kit is labeled.

In other embodiments, the protein in the kit comprising an antigen binding domain that binds CD3 epsilon is labeled.

In other embodiments, the multispecific proteins comprising an antigen-binding domain that binds CD3 epsilon in the kit are labeled.

In other embodiments, the kit comprises an antigen binding domain that binds CD3 epsilon comprising:

VH of SEQ ID NO. 55 and VL of SEQ ID NO. 59;

VH of SEQ ID NO. 55 and VL of SEQ ID NO. 58;

VH of SEQ ID NO. 54 and VL of SEQ ID NO. 56;

VH of SEQ ID NO. 48 and VL of SEQ ID NO. 58;

The VH of SEQ ID NO. 88 and the VL of SEQ ID NO. 58; or alternatively

The VH of SEQ ID NO. 242 and the VL of SEQ ID NO. 58.

In other embodiments, the kit comprises an antigen binding domain that binds CD3 epsilon comprising SEQ ID NO 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or 126.

Method for detecting CD3 epsilon

The invention also provides a method of detecting CD3 epsilon in a sample comprising obtaining a sample, contacting the sample with an antigen binding domain of the present disclosure that binds CD3 epsilon, and detecting CD3 epsilon bound in the sample.

In other embodiments, the sample may be derived from urine, blood, serum, plasma, saliva, ascites fluid, circulating cells, synovial fluid, circulating cells, non-tissue associated cells (i.e., free cells), tissue (e.g., surgically excised tissue, biopsy, including fine needle aspiration tissue), histological preparations, and the like.

The antigen binding domain of the disclosure that binds CD3 epsilon can be detected using known methods. Exemplary methods include directly labeling an antibody with a fluorescent or chemiluminescent label, or a radiolabel, or attaching a readily detectable moiety (such as biotin, an enzyme, or an epitope tag) to an antibody of the invention. Exemplary labels and moieties are ruthenium, ¹¹¹ In-DOTA、 ¹¹¹ In-diethylenetriamine pentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase,Polyhistidine (HIS tag), acridine dye, cyanine dye, fluorone dye, oxazine dye, phenanthridine dye, rhodamine dye, anda dye. />

The antigen binding domains of the present disclosure that bind CD3 epsilon can be used in a variety of assays to detect CD3 epsilon in a sample. Exemplary assays are western blot analysis, radioimmunoassay, surface plasmon resonance, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence Activated Cell Sorting (FACS) or ELISA assays.

Polynucleotides, vectors, and host cells

The present disclosure also provides an isolated polynucleotide encoding any CD3 epsilon binding protein of the present disclosure. CD3 epsilon binding proteins include antigen binding domains that bind CD3 epsilon, proteins comprising antigen binding domains that bind CD3 epsilon, and multispecific proteins comprising antigen binding domains of the present disclosure that bind CD3 epsilon.

The invention also provides an isolated polynucleotide encoding any CD3 epsilon binding protein or fragment thereof.

The invention also provides an isolated polynucleotide encoding a VH of SEQ ID NO. 55, 54 or 48.

The invention also provides an isolated polynucleotide encoding VL of SEQ ID NO. 59, 58 or 56.

The invention also provides an isolated polynucleotide encoding a VH of SEQ ID NO. 55.

The invention also provides an isolated polynucleotide encoding a VH of SEQ ID No. 54.

The invention also provides an isolated polynucleotide encoding a VH of SEQ ID No. 48.

The invention also provides an isolated polynucleotide encoding VL of SEQ ID NO. 59.

The invention also provides an isolated polynucleotide encoding VL of SEQ ID NO. 58.

The invention also provides an isolated polynucleotide encoding VL of SEQ ID NO. 56.

The invention also provides an isolated polynucleotide encoding the VH of SEQ ID NO. 55, 54 or 48 and the VL of SEQ ID NO. 24, 27, 28, 29 or 30.

The invention also provides an isolated polynucleotide encoding:

VH of SEQ ID NO. 55 and VL of SEQ ID NO. 59;

VH of SEQ ID NO. 55 and VL of SEQ ID NO. 58;

VH of SEQ ID NO. 54 and VL of SEQ ID NO. 56;

VH of SEQ ID NO. 48 and VL of SEQ ID NO. 58;

the VH of SEQ ID NO. 88 and the VL of SEQ ID NO. 58; or alternatively

The VH of SEQ ID NO. 242 and the VL of SEQ ID NO. 58.

The invention also provides an isolated polynucleotide encoding a polypeptide of SEQ ID NO. 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125 or 126.

The invention also provides an isolated polynucleotide encoding a polypeptide of SEQ ID NO. 96.

The invention also provides an isolated polynucleotide encoding a polypeptide of SEQ ID NO. 97.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 98.

The invention also provides an isolated polynucleotide encoding a polypeptide of SEQ ID NO. 99.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 100.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 101.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 102.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 103.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 104.

The invention also provides an isolated polynucleotide encoding a polypeptide of SEQ ID NO. 105.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 106.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 107.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 108.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 109.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 110.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 112.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 113.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 114.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 115.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 116.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 117.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 118.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 119.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 120.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 121.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 122.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 123.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 124.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 125.

The invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO. 126.

Some embodiments of the present disclosure also provide an isolated or purified nucleic acid comprising a polynucleotide complementary to a polynucleotide encoding a CD3 epsilon binding protein of the present disclosure or a polynucleotide that hybridizes under stringent conditions to a polynucleotide encoding a CD3 epsilon binding protein of the present disclosure.

Polynucleotide sequences that hybridize under stringent conditions can hybridize under high stringency conditions. By "high stringency conditions" is meant that the polynucleotide hybridizes specifically to a target sequence (the nucleotide sequence of any of the nucleic acids described herein) in a detectably greater amount than does nonspecific hybridization. High stringency conditions include conditions that distinguish polynucleotides having precisely complementary sequences, or polynucleotides containing only a small number of discrete mismatches, from random sequences that have exactly a small number of small regions (e.g., 3 to 12 bases) that match the nucleotide sequence. Such small complementary regions are easier to melt than full-length complementary sequences of 14 to 17 or more bases, and high stringency hybridization allows them to be readily distinguished. Relatively high stringency conditions will include, for example, low salt conditions and/or high temperature conditions, such as those provided by about 0.02M to 0.1M NaCl, or equivalent, at a temperature of about 50 ℃ to 70 ℃. Such high stringency conditions allow for little, if any, mismatch between the nucleotide sequence and the template or target strand. It is believed that conditions may be more stringent by the addition of incremental formamide.

The polynucleotide sequences of the present disclosure are operably linked to one or more regulatory elements, such as promoters or enhancers that allow for the expression of the nucleotide sequence in the intended host cell. The polynucleotide may be a cDNA. The promoter may be a strong promoter, a weak promoter, a tissue specific promoter, an inducible promoter or a development specific promoter. Exemplary promoters that may be used are hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin, human hemoglobin, human muscle creatine, and the like. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use with the embodiments. Such viral promoters include the Cytomegalovirus (CMV) immediate early promoter, the SV40 early and late promoters, the Mouse Mammary Tumor Virus (MMTV) promoter, the Long Terminal Repeat (LTR) of the marone leukemia virus, the Human Immunodeficiency Virus (HIV), the Epstein Barr Virus (EBV), the Rous Sarcoma Virus (RSV) and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Inducible promoters such as metallothionein promoters, tetracycline-inducible promoters, doxycycline-inducible promoters, promoters containing one or more interferon-stimulated response elements (ISRE), such as protein kinase R2 ',5' -oligoadenylate synthetase, mx genes, ADAR1, and the like, may also be used.

The invention also provides a vector comprising a polynucleotide of the invention. The present disclosure also provides an expression vector comprising a polynucleotide of the present invention. Such vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon-based vectors or any other suitable vector for introducing the synthetic polynucleotide of the invention into a given organism or genetic background by any means. The polynucleotide encoding the CD3 epsilon binding protein of the present disclosure may be operably linked to control sequences in an expression vector that ensure expression of the CD3 epsilon binding protein. Such regulatory elements may comprise a transcriptional promoter, sequences encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The expression vector may also contain one or more non-transcriptional elements such as an origin of replication, suitable promoters and enhancers linked to the gene to be expressed, other 5 'or 3' flanking non-transcribed sequences, 5 'or 3' untranslated sequences such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, or transcription termination sequences. An origin of replication conferring replication in a host may also be incorporated.

The expression vector may comprise naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. Non-naturally occurring or altered nucleotides or internucleotide linkages do not interfere with transcription or replication of the vector.

Once incorporated into an appropriate host, the host is maintained under conditions suitable for high level expression of the CD3 epsilon binding protein of the present disclosure encoded by the incorporated polynucleotide. Transcriptional and translational control sequences in expression vectors for transformation of vertebrate cells can be provided by viral sources. Exemplary vectors can be constructed as described by Okayama and Berg,3mol.cell.biol.280 (1983).

Vectors of the present disclosure may also contain one or more Internal Ribosome Entry Sites (IRES). The inclusion of IRES sequences in fusion vectors may be advantageous in enhancing the expression of some proteins. In other embodiments, the vector system will include one or more polyadenylation sites (e.g., SV 40), which may be upstream or downstream of any of the nucleic acid sequences described above. The vector components may be linked serially, or arranged in a manner that provides optimal spacing for expression of the gene product (i.e., by introducing "spacer" nucleotides between ORFs), or positioned in another manner. Regulatory elements such as IRES motifs may also be arranged to provide optimal spacing for expression.

The carrier of the present disclosure may be circular or linear. They can be prepared to comprise replication systems that function in prokaryotic or eukaryotic host cells. Replication systems may be derived from, for example, colE1, SV40, 2 μ plasmids, λ, bovine papilloma virus, and the like.

Recombinant expression vectors can be designed for transient expression, for stable expression, or for both. Furthermore, recombinant expression vectors can be prepared for constitutive expression or for inducible expression.

In addition, recombinant expression vectors may be prepared to include suicide genes. As used herein, the term "suicide gene" refers to a gene that causes death of cells expressing the suicide gene. Suicide genes may be genes that confer sensitivity to an agent, such as a drug, on a cell expressing the gene and cause cell death when the cell is contacted with or exposed to the agent. Suicide genes are known in the art and include, for example, the Herpes Simplex Virus (HSV) Thymidine Kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase. The vector may also contain a selectable marker, which is well known in the art. Selectable markers include positive and negative selectable markers. Marker genes include biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.), complementation to provide prototrophy in an auxotrophic host, and the like. Exemplary marker genes include antibiotic resistance genes (e.g., neomycin resistance gene, hygromycin resistance gene, kanamycin resistance gene, tetracycline resistance gene, penicillin resistance gene, histidinol x resistance gene), glutamine synthase gene, HSV-TK derivatives for ganciclovir selection, or bacterial purine nucleoside phosphorylase genes for 6-methylpurine selection (Gadi et al, 7Gene Ther.1738-1743 (2000)). The nucleic acid sequence encoding the selectable marker or cloning site may be upstream or downstream of the nucleic acid sequence encoding the polypeptide or cloning site of interest.

Exemplary vectors that may be used are bacteria: pBs, phagescript, psiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, la Jolla, calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 (Pharmacia, uppsala, sweden). And (3) eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG and pSVL (Pharmacia), pEE6.4 (Lonza) and pEE12.4 (Lonza). Additional vectors include pUC series (Fermentas Life Sciences, glen Burnie, md.), pBluescript series (Stratagene, laJolla, calif.), pET series (Novagen, madison, wis.), pGEX series (Pharmacia Biotech, uppsala, sweden) and pEX series (Clontech, palo Alto, calif.). Phage vectors such as λgt10, λgt11, λembl4, and λnm1149, λ ZapII (Stratagene) can be used. Exemplary plant expression vectors include pBI01, pBI01.2, pBI121, pBI101.3, and pBIN19 (Clontech). Exemplary animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The expression vector may be a viral vector, such as a retroviral vector, e.g., a gamma retroviral vector.

In other embodiments, the vector comprises a polynucleotide encoding a VH of SEQ ID NO. 55.

In other embodiments, the vector comprises a polynucleotide encoding a VH of SEQ ID NO. 54.

In other embodiments, the vector comprises a polynucleotide encoding a VH of SEQ ID NO. 48.

In other embodiments, the vector comprises a polynucleotide encoding VL of SEQ ID NO. 59.

In other embodiments, the vector comprises a polynucleotide encoding VL of SEQ ID NO. 58.

In other embodiments, the vector comprises a polynucleotide encoding VL of SEQ ID NO. 56.

In other embodiments, the vector comprises a polynucleotide encoding the VH of SEQ ID NO. 55, 54 or 48 and the VL of SEQ ID NO. 59, 58 or 56.

In other embodiments, the vector comprises a polynucleotide encoding:

VH of SEQ ID NO. 55 and VL of SEQ ID NO. 59;

VH of SEQ ID NO. 55 and VL of SEQ ID NO. 58;

VH of SEQ ID NO. 54 and VL of SEQ ID NO. 56;

VH of SEQ ID NO. 48 and VL of SEQ ID NO. 58;

the VH of SEQ ID NO. 88 and the VL of SEQ ID NO. 58; or alternatively

The VH of SEQ ID NO. 242 and the VL of SEQ ID NO. 58.

In other embodiments, the vector comprises a polynucleotide encoding a polypeptide of SEQ ID NO 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125 or 126.

In other embodiments, the vector comprises a polynucleotide encoding a polypeptide of SEQ ID NO. 96.

In other embodiments, the vector comprises a polynucleotide encoding a polypeptide of SEQ ID NO. 97.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 98.

In other embodiments, the vector comprises a polynucleotide encoding a polypeptide of SEQ ID NO. 99.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 100.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 101.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 102.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 103.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 104.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 105.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 106.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 107.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 108.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 109.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 110.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 112.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 113.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 114.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 115.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 116.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 117.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 118.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 119.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 120.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 121.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 122.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 123.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 124.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 125.

In other embodiments, the vector comprises a polynucleotide encoding the polypeptide of SEQ ID NO. 126.

The invention also provides a host cell comprising one or more vectors of the invention. "host cell" refers to a cell into which a vector has been introduced. It should be understood that the term host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell, and also to stable cell lines produced by the particular subject cell. Such progeny may differ from the parent cell, as some modification may occur in the progeny due to mutation or due to environmental effects, but are still included within the scope of the term "host cell" as used herein. Such host cells may be eukaryotic, prokaryotic, plant or archaeal. Examples of prokaryotic host cells are E.coli (Escherichia coli), bacillus species (bacillus) such as B.subtilis (Bacillus subtilis) and other Enterobacteriaceae (such as Salmonella (Salmonella), serratia (Serratia)) and various Pseudomonas species. Other microorganisms such as yeast may also be used for expression. Saccharomyces (e.g., saccharomyces cerevisiae) and Pichia are examples of suitable yeast host cells. Exemplary eukaryotic cells may be of mammalian, insect, avian, or other animal origin. Mammalian eukaryotic cells include immortalized cell lines such as hybridoma or myeloma cell lines, such as SP2/0 (American type culture Collection (ATCC), manassas, VA, CRL-1581), NS0 (European Collection of cell cultures (ECACC), salisbury, wiltshire, UK, ECACC No. 8510503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murine cell lines. An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196). Other useful cell lines include those derived from Chinese Hamster Ovary (CHO) cells, such as CHO-K1SV (Lonza Biologics, walkersville, MD), CHO-K1 (ATCC CRL-61), or DG44.

The present disclosure also provides a method of producing a CD3 epsilon binding protein of the present disclosure, comprising culturing a host cell of the present disclosure under conditions such that the CD3 epsilon binding protein is expressed, and recovering the CD3 epsilon binding protein produced by the host cell. Methods for preparing proteins and purifying proteins are known. Once synthesized (chemically or recombinantly), the CD3 epsilon binding protein may be purified according to standard procedures, including ammonium sulfate precipitation, affinity chromatography columns, column chromatography, high Performance Liquid Chromatography (HPLC) purification, gel electrophoresis, and the like (see generally Scopes, protein Purification (Springer-Verlag, n.y., (1982)). Subject proteins may be substantially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or at least about 98% to 99% pure, or more pure, e.g., free of contaminants, such as cell debris, macromolecules other than subject proteins, and the like.

The polynucleotides encoding the CD3 epsilon binding proteins of the present disclosure can be incorporated into vectors using standard molecular biological methods. Host cell transformation, culture, antibody expression and purification are accomplished using well known methods.

Modified nucleotides may be used to generate polynucleotides of the present disclosure. Exemplary modified nucleotides are 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxymethyl) uracil, carboxymethyl aminomethyl-2-thiouridine, 5-carboxymethyl aminomethyluracil, dihydrouracil, N ⁶ -substituted adenine, 7-methylguanine, 5-methylaminomethyl uracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosyl quinoline, 5 "-methoxycarboxymethyl uracil, 5-methoxyuracil, 2-methylthio-N ⁶ -isopentenyl adenine, uracil-5-oxyacetic acid (v), huai Dingyang glycoside (wybutoxosine), pseudouracil, pigtail glycoside, beta-D-galactosyl pigtail glycoside, inosine, N ⁶ -isopentenyl adenine, 1-methylguanine, 1-methyl inosine, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, 2-thiocytosine, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetate methyl ester, 3- (3-amino-3-N-2-carboxypropyl) uracil and 2, 6-diaminopurine.

Pharmaceutical composition/administration

The present disclosure also provides a pharmaceutical composition comprising a CD3 epsilon binding protein of the present disclosure and a pharmaceutically acceptable carrier.

The present disclosure also provides a pharmaceutical composition comprising the antigen binding domain of the present disclosure that binds CD3 epsilon and a pharmaceutically acceptable carrier.

The present disclosure also provides a pharmaceutical composition comprising a protein comprising an antigen binding domain of the present disclosure that binds CD3 epsilon and a pharmaceutically acceptable carrier.

The present disclosure also provides a pharmaceutical composition comprising a multispecific protein comprising an antigen-binding domain of the present disclosure that binds CD3 epsilon and a pharmaceutically acceptable carrier.

The present disclosure also provides a pharmaceutical composition comprising a multispecific protein comprising an antigen-binding domain of the present disclosure that binds CD3 epsilon and an antigen-binding domain that binds a tumor antigen, and a pharmaceutically acceptable carrier.

For therapeutic use, the CD3 epsilon binding proteins of the present disclosure can be prepared in the form of pharmaceutical compositions containing an effective amount of the antibody as an active ingredient in a pharmaceutically acceptable carrier. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional well-known sterilization techniques, such as filtration. These compositions may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizers, thickeners, lubricants, colorants, and the like.

The term "pharmaceutically acceptable" as used herein with respect to a pharmaceutical composition means a substance approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals and/or humans.

Therapeutic methods and uses

The present disclosure also provides a bispecific protein or a multispecific protein comprising a first antigen-binding domain that specifically binds CD3 epsilon and a second antigen-binding domain that specifically binds a second antigen of the present disclosure for use in therapy.

The present disclosure also provides a bispecific protein or a multispecific protein comprising a first antigen-binding domain that specifically binds to CD3 epsilon and a second antigen-binding domain that specifically binds to a second antigen of the present disclosure for use in treating a cell proliferative disorder.

The present disclosure also provides a bispecific protein or a multispecific protein for killing cancer cells, the bispecific protein or multispecific protein comprising a first antigen-binding domain that specifically binds CD3 epsilon and a second antigen-binding domain that specifically binds a second antigen of the present disclosure.

The present disclosure also provides a bispecific protein or a multispecific protein comprising a first antigen-binding domain that specifically binds to CD3 epsilon and a second antigen-binding domain that specifically binds to a second antigen of the present disclosure for use in the manufacture of a medicament for killing cancer cells.

The present disclosure also provides a bispecific protein or multispecific protein for redirecting cytolytic T cells, the bispecific protein or multispecific protein comprising a first antigen-binding domain that specifically binds to CD3 epsilon and a second antigen-binding domain that specifically binds to a second antigen of the present disclosure.

The present disclosure also provides a bispecific protein or multispecific protein comprising a first antigen-binding domain that specifically binds to CD3 epsilon and a second antigen-binding domain that specifically binds to a second antigen of the present disclosure for use in the manufacture of a medicament for redirecting cytolytic T cells.

The present disclosure also provides a bispecific protein or a multispecific protein for redirecting cytolytic T cells in a tumor microenvironment, the bispecific protein or multispecific protein comprising a first antigen-binding domain that specifically binds to CD3 epsilon and a second antigen-binding domain that specifically binds to a second antigen of the present disclosure.

The present disclosure also provides a bispecific protein or multispecific protein comprising a first antigen-binding domain that specifically binds to CD3 epsilon and a second antigen-binding domain that specifically binds to a second antigen of the present disclosure for use in the manufacture of a medicament for redirecting cytolytic T cells in a tumor microenvironment.

The present disclosure also provides a bispecific protein or a multispecific protein comprising a first antigen-binding domain that specifically binds CD3 epsilon and a second antigen-binding domain that specifically binds a second antigen of the present disclosure for use in treating cancer.

The present disclosure also provides a bispecific protein or a multispecific protein comprising a first antigen-binding domain that specifically binds CD3 epsilon and a second antigen-binding domain that specifically binds a second antigen of the present disclosure for use in the manufacture of a medicament for treating cancer.

In one aspect, the present disclosure relates generally to treatment of a subject at risk of developing cancer. The invention also includes the treatment of the following malignancies: wherein chemotherapy and/or immunotherapy results in significant immunosuppression in the subject, thereby increasing the risk of the subject developing cancer.

The present disclosure also provides a method of treating a non-cancerous condition in a subject at risk of developing a cancerous condition comprising administering to the subject the antigen binding domain of the present disclosure that binds CD3 epsilon to treat the non-cancerous condition.

The present disclosure also provides a method of treating a non-cancerous condition in a subject at risk of developing a cancerous condition comprising administering to the subject a protein of the present disclosure comprising an antigen binding domain that binds CD3 epsilon to treat the non-cancerous condition.

The present disclosure also provides a method of treating a non-cancerous condition in a subject at risk of developing a cancerous condition comprising administering to the subject a multispecific protein of the present disclosure comprising an antigen binding domain that binds CD3 epsilon to treat the non-cancerous condition.

The present disclosure also provides a method of treating a non-cancerous condition in a subject at risk of developing a cancerous condition comprising administering to the subject an immunoconjugate of the disclosure to treat the non-cancerous condition.

The present disclosure also provides a method of treating a non-cancerous condition in a subject at risk of developing a cancerous condition comprising administering to the subject a pharmaceutical composition of the present disclosure to treat the non-cancerous condition.

The present disclosure also provides a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a multispecific protein comprising an antigen-binding domain that binds CD3 epsilon to treat cancer, wherein the antigen-binding domain that binds CD3 epsilon comprises:

VH of SEQ ID NO. 55 and VL of SEQ ID NO. 59;

VH of SEQ ID NO. 55 and VL of SEQ ID NO. 58;

VH of SEQ ID NO. 54 and VL of SEQ ID NO. 56;

VH of SEQ ID NO. 48 and VL of SEQ ID NO. 58;

the VH of SEQ ID NO. 88 and the VL of SEQ ID NO. 58; or alternatively

The VH of SEQ ID NO. 242 and the VL of SEQ ID NO. 58.

The present disclosure also provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a multispecific protein comprising an antigen-binding domain that binds CD3 epsilon to treat cancer, wherein the antigen-binding domain that binds CD3 epsilon comprises the amino acid sequence of SEQ ID NOs 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or 126.

Another aspect of the present disclosure is a method of treating a cell proliferative disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a bispecific protein or multispecific protein comprising a first antigen-binding domain that specifically binds to CD3 epsilon and a second antigen-binding domain that specifically binds to a second antigen of the present disclosure. In other embodiments, a bispecific protein or a multispecific protein comprising a first antigen-binding domain that specifically binds to CD3 epsilon and a second antigen-binding domain that specifically binds to a second antigen of the present disclosure is administered to a subject.

In any of the foregoing uses or methods, the cell proliferative disorder is cancer. In other embodiments, the cancer is selected from the group consisting of: esophageal cancer, gastric cancer, small intestine cancer, large intestine cancer, colorectal cancer, breast cancer, non-small cell lung cancer, non-hodgkin lymphoma (NHL), B-cell lymphoma, B-cell leukemia, multiple myeloma, renal cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, germinal center B-like (GCB) DLBCL, activated B-like (ABC) DLBCL, follicular Lymphoma (FL), mantle Cell Lymphoma (MCL), acute Myelogenous Leukemia (AML), chronic Lymphocytic Leukemia (CLL), marginal Zone Lymphoma (MZL), small Lymphocytic Leukemia (SLL), lymphoplasmacytic Lymphoma (LL), megaloblastic (WM), central Nervous System Lymphoma (CNSL), burkitt Lymphoma (BL) B-cell pre-lymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma/leukemia, unclassified splenic diffuse small-cell erythromyelomas, variant-type hairy cell leukemia, megaloblastic, heavy chain disease, plasma cell myeloma, bone solitary plasmacytoma, bone exoplasmacytoma, mucosa-associated lymphoid tissue lymph node exoedge zone lymphoma (MALT lymphoma), lymph node marginal zone lymphoma, childhood node endoedge zone lymphoma, childhood follicular lymphoma, primary skin follicular central lymphoma, T cell/tissue cell-enriched large B cell lymphoma, primary central nervous system DLBCL, primary skin DLBCL-leg, elderly EBV positive DLBCL, chronic inflammation-associated DLBCL, lymphomatoid granulomatosis, primary mediastinal (thymus) large B-cell lymphomas. Intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablasts lymphoma, HHV 8-related multicenter Castleman disease-induced large B-cell lymphoma, primary exudative lymphoma: unclassified B-cell lymphomas characterized by a range between diffuse large B-cell lymphomas and burkitt's lymphomas and unclassified B-cell lymphomas characterized by a range between diffuse large B-cell lymphomas, classical hodgkin lymphomas and light chain amyloidosis.

In other embodiments, the cancer is esophageal cancer. In other embodiments, the cancer is an adenocarcinoma, such as a metastatic adenocarcinoma (e.g., colorectal adenocarcinoma, gastric adenocarcinoma, or pancreatic carcinoma).

In another aspect, the disclosure features a kit that includes: (a) A composition comprising any of the foregoing bispecific or multispecific proteins comprising a first antigen-binding domain that specifically binds to CD3 epsilon and a second antigen-binding domain that specifically binds to a second antigen of the present disclosure, and (b) package instructions comprising instructions for administering the composition to a subject to treat or delay progression of a cell proliferative disorder.

In any of the foregoing uses or methods, the subject may be a human.

Combination therapy

The CD3 epsilon binding proteins of the present disclosure can be administered in combination with at least one additional therapeutic agent.

In other embodiments, delivery of one treatment is still ongoing at the beginning of delivery of a second treatment, such that there is overlap in administration. This is sometimes referred to herein as "simultaneous" or "simultaneous delivery. In other embodiments, the delivery of one therapy ends before the delivery of another therapy begins. In some embodiments of either case, the treatment is more effective due to the combined administration. For example, the second treatment is more effective than a similar situation observed when the second treatment is administered in the absence of the first treatment, or when the first treatment is used, e.g., an equivalent effect is observed when less of the second treatment is used, or the second treatment reduces symptoms to a greater extent. In other embodiments, the delivery results in a reduction in symptoms or other parameters associated with the disorder to a greater extent than would be observed if one treatment were delivered without the presence of the other. The delivery may be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered.

The CD3 epsilon binding protein and at least one additional therapeutic agent described herein may be administered simultaneously, in the same or separate compositions, or sequentially. For sequential administration, the CD3 epsilon binding protein described herein may be administered first, additional agents may be administered second, or the order of administration may be reversed.

Description of the embodiments：

The present invention provides the following non-limiting embodiments.

1. An isolated protein comprising an antigen binding domain that binds to cluster 3 epsilon (CD 3 epsilon), wherein the antigen binding domain that binds to CD3 epsilon comprises:

HCDR1, HCDR2 and HCDR3 of VH of SEQ ID No. 55, LCDR1, LCDR2 and LCDR3 of VL of SEQ ID No. 58;

HCDR1, HCDR2 and HCDR3 of VH of SEQ ID No. 54, LCDR1, LCDR2 and LCDR3 of VL of SEQ ID No. 56; or alternatively

HCDR1, HCDR2 and HCDR3 of VH of SEQ ID No. 48, LCDR1, LCDR2 and LCDR3 of VL of SEQ ID No. 58;

2. An isolated protein comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 70, 71, 86, 79, 80 and 81 respectively.

3. The isolated protein according to embodiment 1 or 2, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as follows:

a. SEQ ID NOs 70, 71, 72, 79, 80 and 81, respectively;

b. 70, 71, 87, 79, 80 and 81 respectively; or alternatively

c. SEQ ID NOS 70, 71, 90, 79, 80 and 81, respectively.

4. The isolated protein of embodiments 1-3, wherein the antigen binding domain that binds CD3 epsilon is an scFv, (scFv) 2, fv, fab, F (ab') 2, fd, dAb, or VHH.

5. The isolated protein of embodiment 4, wherein said antigen binding domain that binds CD3 epsilon is a Fab.

6. The isolated protein of embodiment 4, wherein the antigen binding domain that binds CD3 epsilon is a scFv.

7. The isolated protein of embodiment 6, wherein the scFv comprises VH, first linker (L1) and VL (VH-L1-VL) or comprises VL, L1 and VH (VL-L1-VH) from N-terminus to C-terminus.

8. The isolated protein of embodiment 7, wherein the L1 comprises

a. About 5 to 50 amino acids;

b. About 5 to 40 amino acids;

c. about 10 to 30 amino acids; or alternatively

d. About 10 to 20 amino acids.

9. The isolated protein of embodiment 7, wherein said L1 comprises the amino acid sequence of SEQ ID NOS 3 to 36.

10. The isolated protein of embodiment 9, wherein said L1 comprises the amino acid sequence of SEQ ID NO. 3.

11. The isolated protein of any one of embodiments 1-10, wherein the antigen binding domain that binds CD3 epsilon comprises the VH of SEQ ID No. 55, 54 or 48, and the VL of SEQ ID No. 59, 58 or 56.

12. The isolated protein of embodiment 11, wherein the antigen binding domain that binds CD3 epsilon comprises:

a VH of SEQ ID NO. 55 and VL of SEQ ID NO. 59;

VH of SEQ ID NO. 55 and VL of SEQ ID NO. 58;

VH of SEQ ID NO. 54 and VL of SEQ ID NO. 56;

the VH of SEQ ID NO. 48 and the VL of SEQ ID NO. 58;

e.VH of SEQ ID NO. 88 and VL of SEQ ID NO. 58; or alternatively

VH of SEQ ID NO:242 and VL of SEQ ID NO: 58.

13. The isolated protein of any of embodiments 1-12, wherein the antigen binding domain that binds CD3 epsilon comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 96-126.

14. The isolated protein of any one of embodiments 1-13, wherein the isolated protein is a multi-specific protein.

15. The isolated protein of embodiment 14, wherein the multispecific protein is a bispecific protein.

16. The isolated protein of embodiment 14, wherein the multispecific protein is a trispecific protein.

17. The isolated protein of any of embodiments 1-16, further comprising an immunoglobulin (Ig) constant region or a fragment of the Ig constant region.

18. The isolated protein of embodiment 17, wherein the fragment of the Ig constant region comprises an Fc region.

19. The isolated protein of embodiment 17, wherein the fragment of the Ig constant region comprises a CH2 domain.

20. The isolated protein of embodiment 17, wherein the fragment of the Ig constant region comprises a CH3 domain.

21. The isolated protein of embodiment 17, wherein the fragment of the Ig constant region comprises a CH2 domain and a CH3 domain.

22. The isolated protein of embodiment 17, wherein the fragment of the Ig constant region comprises at least a portion of a hinge, a CH2 domain, and a CH3 domain.

23. The isolated protein of embodiment 17, wherein the fragment of the Ig constant region comprises a hinge, a CH2 domain, and a CH3 domain.

24. The isolated protein of any of embodiments 17-24, wherein the antigen binding domain that binds CD3 epsilon is conjugated to the N-terminus of the Ig constant region or the fragment of the Ig constant region.

25. The isolated protein of any of embodiments 17-24, wherein the antigen binding domain that binds CD3 epsilon is conjugated to the C-terminus of the Ig constant region or the fragment of the Ig constant region.

26. The isolated protein of any of embodiments 17-24, wherein the antigen binding domain that binds CD3 epsilon is conjugated to the Ig constant region or the fragment of the Ig constant region via a second linker (L2).

27. The isolated protein of embodiment 35, wherein said L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 3 to 36.

28. The isolated protein of any of embodiments 14-27, wherein the multispecific protein comprises an antigen-binding domain that binds an antigen that is not CD3 epsilon.

29. The multispecific antibody of embodiments 14-28, wherein the cell antigen is a tumor-associated antigen.

30. The isolated protein of any of embodiments 14-29, wherein the Ig constant region or the fragment of the Ig constant region is an IgG1, igG2, igG3, or IgG4 isotype.

31. The isolated protein of any of embodiments 1-30, wherein the Ig constant region or the fragment of the Ig constant region comprises at least one mutation that causes a reduction in binding of the protein to an fcγ receptor (fcγr).

32. The isolated protein of embodiment 31, wherein the at least one mutation that causes reduced binding of the protein to the fcγr is selected from the group consisting of: F234A/L235A, L A/L235A, L A/L235A/D265S, V234A/G237A/P238S/H268A/V309L/A330S/P331S, F A/L235A, S P/F234A/L235A, N297A, V A/G237A, K T/E233P/L234V/L235A/G236-deletion/A327G/P331A/D365E/L358M, H268Q/V309L/A330S/P331S, S E/L328F, L F/L235E/D265A, L A/L235A/G237A/P238S/H268A/A330S/P331S, S P/F234A/L235A/G237A/P238S and S228P/F234A/L235A/G236-deletion/G237A/P238S, wherein residue numbering is performed according to the EU index.

33. The isolated protein of any of embodiments 31-32, wherein the fcγr is fcγri, fcγriia, fcγriib, or fcγriii, or any combination thereof.

34. The isolated protein of any of embodiments 14-33, wherein the protein comprises at least one mutation in the CH3 domain of the Ig constant region.

35. The isolated protein of embodiment 34, wherein the at least one mutation in the CH3 domain of the Ig constant region is selected from the group consisting of: T350V, L351Y, F A, Y407V, T366Y, T366W, T366L, T L, F405W, T394W, K392L, T394S, T394W, Y407T, Y A, T S/L368A/Y407V, L Y/F405A/Y407V, T I/K392M/T394W, F A/Y407V, T366L/K392M/T394W, T L/K392L/T394W, L57351Y/Y407A, L Y/Y407V, T A/K409F, T366V/K409F, T366A/K409F, T V/L351Y/F405A/Y407V and T350V/T366L/K392L/T394W, wherein residue numbering is according to EU index.

36. A pharmaceutical composition comprising the isolated protein of any one of embodiments 1-35, and a pharmaceutically acceptable carrier.

37. A polynucleotide encoding the isolated protein of any one of embodiments 1 to 35.

38. A vector comprising the polynucleotide of embodiment 35.

39. A host cell comprising the vector of embodiment 38.

40. A method of producing the isolated protein of any one of embodiments 1 to 35, comprising: culturing the host cell of embodiment 39 under conditions such that the protein is expressed, and recovering the protein produced by the host cell.

41. A method of treating cancer in a subject comprising administering to the subject in need thereof a therapeutically effective amount of the isolated protein of any one of embodiments 1 to 35 to treat the cancer.

42. An anti-idiotype antibody that binds to the isolated protein of any one of embodiments 1-35.

43. The isolated protein of any of embodiments 1-35, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 127-157.

44. The isolated protein of any of embodiments 1-35, comprising an antibody heavy chain of SEQ ID NO:224 and an antibody light chain of SEQ ID NO: 226.

Examples

Example 1 generation and characterization of anti-CD 3 mAb

These experiments were performed using a publicly available mouse Cris7 antibody specific for human CD3 epsilon (Alberola-Ila, J. Et al Stimulation through the TCR/CD3 complex up-regulatory the CD2 surface expression on human T lymphocytes.J Immunol 146,1085-1092 (1991)). The VH and VL sequences of Cris-7 are shown below.

Cris-7 VH(SEQ ID NO:37)：

QVQLQQSGAELARPGASVKMSCKASGYTFTRSTMHWVKQRPGQGLEWIGYINPSSAYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCASPQVHYDYNGFPYWGQGTLVTVSA

Cris-7 VL(SEQ ID NO:38)：

QVVLTQSPAIMSAFPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDSSKLASGVPARFSGSGSGTSYSLTISSMETEDAATYYCQQWSRNPPTFGGGTKLQIT

Humanization of CD3 binding Domain and scFv formatting

Evaluation of optimal germline sequences

Murine Cris-7 is humanized as a single chain fragment variable domain (scFv). To find the most thermostable combination of human germline receptor HC and LC pairs for scFv format matching to Cris7, two human heavy chain variable domain (Hv) germline sequences and two human light chain variable domain (Lv) germline sequences were selected for antibody humanization: IGHV1-69 x 02-IGHJ1-01 and IGHV5-10-1 x 01-IIGHJ1-01 for Hv, and IGKV3-11 x 02-IGKJ4-01 or IGKV1-39 x 01-IGKJ4-01 lines for Lv (search from Internet: < URL: http:// www.imgt.org/vquest/refseqh.html >). CDR-grafted sequences with limited back mutations were generated to enhance stability (see table 4 below). These CDR-grafted v regions were then expressed in the form of scFv in e.coli in the orientation of heavy chain-linker-light chain (HL) and light chain-linker-heavy chain (LH). As described below, the matrix of Hv and Lv pairings was evaluated in scFv format as either Lv followed by Hv or Hv followed by Lv with a flexible linker between these variable domains. CD3B1127 and CD3B1128 constitute murine VH and VL sequences (table 4). There are two main conclusions about this experiment. First, in all cases, the Cris-7 derived scFv molecules showed significantly stronger binding to recombinant CD3 (TRCW 5, SEQ ID NO: 39) when in the HL orientation than in the LH orientation, based mainly on a higher maximum signal, as determined by ELISA. Second, the graft construct containing limited back mutations in the heavy chain-light chain orientation for IGHV1-69 x 02-IGHJ1-01 heavy chain and IGKV3-11 x 02-IGKJ4-01 light chain lines showed optimal expression, binding profile and potential differentiation, and was therefore selected for humanization. (FIG. 1, table 4 and Table 5).

TABLE 4 amino acid sequences of the graft sequences which contain limited back mutations。

* HL-VH-linker-VL; LH-VL-linker-VH

TABLE 5 determination of EC50 (nM) of binding of CD 3-specific variants to recombinant CD3 using ELISA。

Proteins	EC50
		CD3B1129	0.7124
CD3B1130	0.7465
		CD3B1131	1.137
CD3B1132	About 1.101
		CD3B1133	0.9583
CD3B1134	About 0.006296
		CD3B1135	1.036
CD3B1136
		CD3B1127	About 0.3972
CD3B1128	About 0.4369
		F5 HL	About 0.005701
Culture medium

Human framework optimization in IGHV1-69 x 02-IGHJ1-01 and IGKV3-11 x 02-IGKJ4-01 lines

As described above, since IGHV1-69 x 02-IGHJ1-01 and IGKV3-11 x 02-IGKJ4-01 germline grafted sequences (CD 3B 1130) showed enhanced binding compared to the murine parent and represent the most similar human germline to the murine parent, human framework adaptation was performed starting from this CDR grafted sequence. Starting from this sequence, several sites in VH that may affect molecular stability were identified, and these sites were selected for library-based mouse reverse mutagenesis (table 6). In one VH library, mutations were made at 4 sites in the binary library (M48I, A60N, V a and I69L-Kabat numbering), and R94 (Kabat numbering) was mutated to S, V, L, K, T, R, I or Y for a total of 128 variants. In the second library, 9 sites in the binary library (K12A, V20M, R38K, M I, A60N, R66, V67A, I69L and R94S-Kabat numbering) were mutated for a total of 512 variants. Such methods are known in the art and are described, for example, in Chiu et al, antibodies 2019,8,55.

TABLE 6 murine Cris-7, human germline VH sequences for humanization, and positions of binary and mutant residues。

Similarly, two libraries of VL sequences were generated by identifying sites that might affect the stability of the molecule. In library 1, no modifications were made to LC. In library 2, 11 loci were selected in binary fashion for mouse reverse mutagenesis (L11M, L13A, A19V, L M, Q42T, A43S, L46R, L47W, I V, F71Y and L78M) for a total of 2048 variants (table 7).

TABLE 7 murine Cris-7, human germline VL sequences for humanization, and positions of binary and mutant residues。

Reverse mutation library is generated by molecular biology techniques known in the art (Thomas s. Et al DNA library construction using Gibson)Nat Methods, p.i-ii, 11 months of 2015). Different acceptor lines were paired with the opposite murine parent chain. Thus, only one strand at a time is the human framework equipped with potential back mutations.

Briefly, DNA was transformed into an e.coli expression vector to generate scFv molecules with a C-terminal HA tag, and then cells were plated on 2xYT/Carb/2% glucose and grown overnight at 37 ℃. Colonies were picked, 50. Mu.l of overnight grown culture was transferred to a new plate containing 500. Mu.l 2XYT/Carb/0.1% glucose, grown for 6 to 7 hours, and then mixed with 50. Mu.l 2XYT medium containing 1 Xcarbenicillin and 12 XIPTG. Cultures were incubated overnight at 30℃with shaking at approximately 600 RPM. Streptavidin-coated plates were combined with 50. Mu.L of biotinylated TRCW5 antigen (CD 3. Delta. Epsilon. -Fc-Avi, SEQ ID NO: 39) at the concentrations indicated in the ELISA plate for 45 min at room temperature with shaking (FIGS. 1, 3, 4), followed by 3 washes with 1 XTBST. Plates were blocked with 200 μl1 XTBE containing 3% milk at room temperature for about 45 min, followed by 3 washes with 1 XTBE. E.coli cultures were harvested by centrifugation at 35000RPM for 10 minutes at 4℃and 50. Mu.L of supernatant was transferred to CD3 coated plates followed by incubation at 4℃for 45 minutes. Plates were washed 3 times with 1 XTBST. Bound scFv was detected with chicken polyclonal anti-HA-HRP (ab 1190) [1:1000] for about 45 minutes at room temperature, followed by detection of luminescence with chemiluminescent substrate.

TRCW5 antigen (CD 3 delta epsilon-Fc-Avi, SEQ ID NO: 39)

FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMGSADDAKKDAAKKDDAKKDDAKKDGSDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGLNDIFEAQKIEWHE

Clones exhibiting stronger binding than the murine parent were selected for sequencing and exposed to titration and heat stress ELISA assays. Briefly, scFv were expressed as above, subjected to thermal stress (heat shock at 60 ℃ for about 10 minutes), followed by ELISA analysis as described above. Briefly, clones containing different combinations of mouse back mutations that showed binding to E.coli supernatant were sequenced to determine which residues at each locus (human or mouse germline residues) were more optimal for maintaining thermostability. The proportion of clones harboring each residue at each position was determined (fig. 2A and 2B). 8 human adaptive heavy chain sequences were selected from the two heavy chain libraries designed, and 8 human adaptive light chain sequences were selected from the single light chain library designed based on retention of more than 70% binding (compared to ELISA binding at room temperature) after being subjected to thermal stress. The sequences of the thermostable humanized Cris-7VH and VL are shown below (Table 8).

TABLE 8 sequences of thermostable humanized Cris-7VH and VL

The 8 new heavy chains and 8 new light chains (shown in table 8) were again matrixed with each other to generate scFv (table 9) and then further exposed to additional assays including titration, thermal stress and cell binding (fig. 3).

TABLE 9 matrixing protein species of thermostable variable domains

For pan T cells (Biological Specialty Corp, cat# 215-02-11) and Jurkat-CD3 negative cellsTIB-153 ^TM ) Cell binding was performed to observe an increase in non-specific binding when subjected to thermal stress. Molecules that show increased binding to negative cell lines upon heat stress are not selected for additional characterization. Binding to recombinant CD3 (TRCW 5, SEQ ID NO: 39) was determined by ELISA as described above. Cell binding was performed by flow cytometry using primary human T cells and Jurkat cells. Briefly, E.coli expresses anti-CD 3 ScFv supernatant at room temperature, or in a sample heat treated at 55, 60 or 65 ℃.

Pan T cells (CD 3 positive) and Jurkat-CD3 negative cells were prepared by: jurkat-CD3 negative cells were stained with CFSE while pan T cells were kept unstained. The Jurkat-CD3 negative cell suspension was resuspended at a density of two tens of millions of cells per 50mL conical tube. Cells were harvested by centrifugation at 400Xg for 5 min and resuspended inDPBSAfter which the cells were washed 2 times in DPBS. Cells were stained with a 1:25,000 dilution with a final dye concentration of 0.02. Mu.M CFSE (2. Mu.L staining solution to 50mL cell suspension). Cells were taken for 10 minutes at room temperature and then centrifuged at 400Xg for 5 minutes. After removing the supernatant, 3mL of HI-FBS was added to the cell pellet, mixed, and centrifuged at 400Xg for 5 minutes. The supernatant was removed and the cells were resuspended in BD staining buffer at a density of 2X 10≡6 cells/mL and incubated in the absence of light on ice or at 4 ℃. Human pan T cells were thawed at 37 ℃ and gently transferred to conical tubes with 15mL of warm or room temperature medium (rpmi+10% FBS). T cells from donor ID number M7348, lot LS 11 62980A had a viability of 97.2%. Cells were harvested by centrifugation at 400Xg for 5 min and resuspended in medium (RPMI+10% HI-FBS). In flowing staining buffer at 2× Pan T cells (unstained) were prepared at a density of 10≡6 cells/mL. Equal volumes of CFSE stained Jurkat-CD3 negative cells and unstained (CD 3 positive) T cells were mixed and plated into assay plates at 50 μl/well. Pure bacterial supernatant samples and control samples (CD 3W36 ScFv) were taken and added to each well in an amount of 50 μl/well, and the plates were incubated at a temperature of 4 ℃ or higher for 1 hour. Cells were harvested by centrifugation at 400Xg for 4 min, 150. Mu.L of staining buffer was added to all wells, followed by centrifugation at 400Xg for 4 min to pellet cells. To each well 150 μl of staining buffer was added followed by centrifugation at 400xg for 4 minutes to pellet the cells. A647 conjugated anti-HIS secondary antibody (diluted 1:100 from stock vials) was prepared at 2. Mu.g/mL in staining buffer and then added to the washed cells in an amount of 50. Mu.L/well, followed by incubation at 4℃for 30 minutes in the absence of light. Then 150. Mu.L of staining buffer was added to all wells and the plate was spun at 400Xg for 4 minutes to pellet the cells. mu.L of IntelliCyt running buffer was added to all wells and the plates were spun at 400Xg for 4 minutes to pellet the cells. Cells were resuspended in 20 to 30 μl of running buffer containing 1:1000 dilution of Sytox Blue dead cell stain and the plates were run on an iQue screen. Briefly, cells were gated on FCS versus SCS plots to eliminate debris. Single cells were gated on SCS-se:Sup>A to SCS-H, cells were first isolated based on CFSE staining on BL1 channel and gated on VL1 channel, and then low/negative stained cells were selected with Sytox Blue viability stain to gate on living cells. Binding of scFv molecules was assessed by geometry on RL1 channels from living cell populations. Data were analyzed in GraphPad Prism. Finally, four humanized matrixed clones exhibited the most desirable properties: the four clones were selected as they remained bound after heat shock at 65 ℃. These clones were designated as CD3B2029, CD3B2030, CD3B2051 and CD3B2089.

The amino acid sequences, DNA sequences, and CDR sequences of VH and VL of CD3B2029, CD3B2030, CD3B2051, and CD3B2089 are shown in tables 10, 11, and 12.

Table 13 shows the thermostability ELISA data for all clones tested in Table 9, which bind to recombinant CD3 (TRCW 5, SEQ ID NO: 39).

Table 14 shows the cell stability ELISA binding data for selected clones.

TABLE 10 VH and VL amino acid sequences for CD3B2029, CD3B2030, CD3B2051 and CD3B2089

TABLE 11 VH and VL DNA sequences of CD3B2029, CD3B2030, CD3B2051 and CD3B2089。

TABLE 12 CDRs of CD3B2029, CD3B2030, CD3B2051 and CD3B2089 using different depictions Amino acid sequence。

TABLE 13 percent binding to recombinant CD3 remaining after heat exposure, determined by ELISA。

Table 14 shows thermal stabilityQualitative cell binding data。

Reducing risk of post-translational modification

The parent molecule was determined to contain a "NG" motif at positions 106 to 107 in CDRH3, where the position numbers counted starting from the N-terminus of the VH of the CD3B2029, CD3B2030, CD3B2051 or CD3B2089 sequence (SEQ ID NOs: 55, 54 or 48). The "NG" motif may potentially risk post-translational modification (PTM), in particular Asn deamidation, leading to loss of activity. To mitigate this risk of PTM, selected humanized variants CD3B2029, CD3B2030, CD3B2051 and CD3B2089 were further mutated at the N106 position, respectively, using molecular biology techniques well known in the art (table 15 and table 16). Position N106 is mutated to one of the following residues: A/G/S/F/E/T/R/V/I/Y/L/P/Q/K. As described above, these new variants were again exposed to various assays, including titration, thermal stress, and cell binding. EC50 values for binding to pan T cells and Jurkat cells as determined by ELISA are shown in table 17, and the binding curves of the CD3B2030 variant to recombinant CD3 (TRCW 5, SEQ ID NO: 39) are shown in fig. 4 as an example. The indicated% retention after heat exposure is shown in table 18. Based on these assays, 4 individual amino acid substitutions at the N-position were selected for further testing. Most mutations at N106 remain bound to some extent and all are considered valuable because they both provide a means of modulating T cell redirection efficiency and can successfully eliminate the risk of deamidation at N106.

Table 15 shows the variant CDR sequences generated using the CD3B2029, CD3B2030, CD3B2051 and CD3B2089 sequences.

Table 16 shows a list of substitutions in the HCDR3 sequences generated using the CD3B2029, CD3B2030, CD3B2051 and CD3B2089 sequences, where the position numbers counted from the N-terminus of the VH of CD3B2029, CD3B2030, CD3B2051 or CD3B2089 (SEQ ID NO:55, 54 or 48).

TABLE 15 CD3B2029, CD3B2030, CD3B2051 and CD3B2089 using Kabat depiction CDR amino acid sequences。

TABLE 16 extraction from HCDR3 sequences generated with the CD3B2029, CD3B2030, CD3B2051 and CD3B2089 sequences Generation, wherein position numbers are from CD3B2029, CD3B2030, CD3B2051 or CD3B2089 (SEQ ID NO:55, 54 or 48) N-terminal start counting of VH。

TABLE 17 EC50 values for humanized Cris-7 variants with reduced N106 PTM。

TABLE 18 thermal stability analysis of PTM-reduced humanized Cris-7 variants。

	% retention at 55 °c	% retention at 60 °c	% retention at 65 °c
				CD3B2030 N106Q	109.6％	97.5％	72.3％
CD3B2030 N106H	103.5％	98.9％	55.7％
				CD3B2030 N106A	111.0％	102.7％	55.6％
CD3B2030 N106R	107.1％	104.2％	53.8％
				CD3B2030	104.4％	102.4％	41.3％
CD3B2030 N106L	107.2％	100.5％	25.6％
				CD3B2030 N106F	106.1％	83.3％	21.8％
CD3B2030 N106K	99.7％	87.2％	7.3％
				CD3B2030 N106G	114.7％	59.8％	1.7％
CD3B2030 N106S	64.6％	19.0％	0.6％
				CD3B2051 N106A	98.9％	97.0％	33.3％
CD3B2051	103.9％	95.0％	10.9％
				CD3B2051 N106H	103.1％	84.2％	9.0％
CD3B2051 N106R	101.2％	74.5％	8.4％
				CD3B2051 N106K	116.5％	91.3％	4.0％
CD3B2051 N106S	100.9％	70.9％	1.5％
				CD3B2051 N106G	98.9％	33.1％	1.5％
CD3B2051 N106V	54.9％	1.4％	0.7％
				CD3B2051 N106E	78.4％	15.4％	0.5％
CD3B2051 N106P	87.7％	32.6％	0.2％
				CD3B2089 N106R	101.6％	103.0％	60.4％
CD3B2089 N106F	112.7％	101.3％	36.2％
				CD3B2089 N106E	113.3％	108.3％	33.2％
CD3B2089	104.5％	109.2％	15.1％
				CD3B2089 N106S	102.3％	90.2％	5.4％
CD3B2089 N106T	108.4％	76.7％	1.4％
				CD3B2089 N106I	40.6％	2.5％	0.9％

Based on ELISA data we selected two substitutions with similar affinity to the parent molecule. For the NG motif we chose to replace the N106 position with Q or a. Furthermore, it is desirable to have potential substitutions that can reduce affinity. For the NG motif, we selected G and S for the N position (based on ELISA data, this moderately reduces affinity). These variants were then formatted as bsAb for further analysis of their ability to mediate cytotoxicity and their biophysical characteristics. Note that the Cris-7 based scFv portion is formatted as bsAb in both LH and HL orientations. LH orientation can furthermore modulate affinity for CD3, thus modulating the efficiency of T cell redirection.

Table 19 shows the sequences of the selected CD 3-specific variants.

TABLE 19 sequences for selection of CD 3-specific variants using the Kabat description。

Epitope identification

The epitope on CD3 was determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS). Antibody clone OKT3 was used as a control for the HDX experiment because its epitope on CD3 ε is known from the crystal structure (PDB ID 1SY 6) (Kjer-Nielsen, L. Et al; proc Natl Acad Sci U S A.101, 7675-7680).

Exchange experiments in HDX-MS exchange reactions were initiated by mixing 10. Mu.L of 10. Mu.M CD3W220 (SEQ ID NO: 5) (consisting of CD 3. Epsilon. Gamma. Fused to a 26-aa linker region fused to the serum albumin domain) with or without a 1.2 molar excess of ligand and 30. Mu.L of H2O or deuterated buffer (20 mM MES, pH 6.4, 95% D2O solution of 150mM NaCl or 95% D2O solution of 20mM Tris, pH 8.4, 150mM NaCl). The reaction mixture was incubated at 1.2℃for 15 seconds, 50 seconds, 150 seconds, 500 seconds or 1,500 seconds. The exchanged solution was quenched by the addition of cooled 40 μl of 8M urea, 1M TCEP (pH 3.0) and immediately analyzed.

CD3W220(CD3εγ-HSA-6xHis)(SEQ ID NO:92)：

QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVGSADDAKKDAAKKDDAKKDDAKKDGSQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGSHHHHHHHH

Procedure for HDX-MS data acquisition. HDX-MS sample preparation was performed using an automated HDx system (LEAP Technologies, morrisville, NC). The column and the pump are; protease, type XIII protease (protease from Aspergillus zoffii, type XIII)/pepsin column (w/w, 1:1;2.1 mm. Times.30 mm) (NovaBioAssays Inc., woburn, mass.); wells, ACQUITY UPLC BEH C VanGuard pre-column (2.1X5 mm) (Waters, milford, mass.), analysis, accumore C18 (2.1X100 mm) (Thermo Fisher Scientific, waltham, mass.); and LC pump, VH-P10-A (Thermo Fisher Scientific). The loading pump (from protease column to trap column) was set with 99% water, 1% acetonitrile and 0.1% formic acid at a flow rate of 600 μl/min. The gradient pump (from trap column to analytical column) was set to gradient from 8% acetonitrile in 0.1% formic acid in water to 28% acetonitrile in 0.1% formic acid in water at a flow rate of 100 μl/min over 20 minutes.

MS data acquisition Using LTQ ^TM Orbitrap Fusion Lumos Mass Spectrometry (Thermo Fisher Scientific) with capillary temperature 275 ℃, resolution 150,000 and mass range (m/z) 300-1,800.

Peptide identification of non-deuterated samples was performed prior to HDX experiments using BioPharma Finder 3.0 (Thermo Fisher Scientific). Centroid values were extracted from MS raw data files of HDX experiments using HDExaminer version 2.5 (Sierra analysis, modesto, CA).

The extracted HDX-MS data was further analyzed in Excel. All exchange time points (at 1.2 ℃ at pH 6.4 or pH 8.4) were converted to equivalent time points at pH 7.4 and 23 ℃ (e.g., 15 seconds at 1.2 ℃ at pH 6.4 equivalent to 0.15 seconds at 23 ℃ at pH 7.4; table 20).

TABLE 20 contrast correction of HDX reaction conditions and exchange time to exchange time at pH 7.4 and 23℃。

Antibody clone OKT3 was used as a control for the HDX experiment, since its epitope on CD3 ε was known from the crystal structure (PDB ID 1SY 6). Consistent with the crystal structure of OKT3 binding to CD3 epsilon, it was found that the epitope of OKT3 consisted of peptides spanning residues 29 to 37, 79 to 84 and 87 to 89. To determine the epitope bound by Cris7b on CD3 ε, a bispecific protein comprising Cris7b-N106Q was used, which was formatted as a Fab (SEQ ID Nos: 93 and 94) and paired with an antigen-specific scFv-Fc arm. This experiment shows that Cris7 interacts with an epitope consisting of peptides spanning residues 33 to 37, 53 to 54 and 79 to 84, which partially overlap with the peptides of OKT3, but also interact with peptides spanning residues 53 to 54, which are unique compared to OKT 3.

Cris7b-N106Q HC，SEQ ID NO 93:

QVQLLQSAAEVKKPGESLKISCKGSGYTFTRSTMHWVRQTPGKGLEWMGYINPSSAYTNYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARPQVHYDYQGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Cris7b-N106Q LC，SEQ ID NO:94

EIVLTQSPSAMSASVGDRVTITCSASSSVSYMNWYQQKPGKVPKRLIYDSSKLASGVPSRFSGSGSGTEYTLTISSLQPEDFATYYCQQWSRNPPTFGQGTMLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

EXAMPLE 2 characterization of bispecific forms of novel CD3 binding Agents。

The VH/VL region of the anti-CD 3 antibody generated in example 1 and the VH/VL region of the anti-BCMA antibody described below were engineered to be bispecific and expressed as IgG1 (table 21).

Engineering CD3 scFv to generate BCMA xCD3 bispecific。

The CD3 VH/VL region was engineered into scFv in a VH-linker-VL (also referred to as "HL") or VL-linker-VH (also referred to as "LH") orientation using the linker of SEQ ID NO:3 (Table 22). The CD 3-binding VH-linker-VL or VL-linker-VH scFv molecules were further engineered into the scFv-hinge-CH 2-CH3 (also known as scFv-Fc) format, which contained an Fc silent mutation (L234A/L235A/D265S) and a T350V/L351Y/F405A/Y407V mutation designed to promote selective heterodimerization (Table 23). The polypeptide of SEQ ID NO. 95 is used as constant domain hinge-CH 2-CH3. The DNA sequences of the anti-CD 3 molecules in scFv format and scFv-hinge-CH 2-CH3 format are shown in table 24.

SEQ ID NO. 95 (huIgG 1-G1 m (17) -hinge-Fc_C220S_AAS_ZWA)

TABLE 21 CD3xBCMA bispecific protein。

HL-VH-linker-VL fused to Fc; LH-and Fc fused VL-linker-VH

TABLE 22 CD3 specific scFv sequences。

TABLE 23 CD3 specific scFv-Fc (scFv-hinge CH2-CH 3) arm。

TABLE 24 DNASEQ ID NO against CD3 scFv and scFy-hinge-CH 2-CH3 (scFv-Fc)

Engineering CD3Fab to generate BCMA xCD3 bispecific

The CD3 specific VH and VL regions were engineered in the VH-CH 1-linker-CH 2-CH3 and VL-CL formats, respectively, and expressed as IgG1. CD 3-specific VH-CH 1-linker-CH 2-CH3 was generated using a polypeptide comprising Fc silent mutation L234A/L235A/D265S and the CH3 mutation T350V/L351Y/F405A/Y407V, designed to promote selective heterodimerization, SEQ ID NO:220 (Table 25).

SEQ ID NO:220(huIgG1_G1m(17)_AAS_ZWA)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

CD 3-specific VL-CL (Table 26) was generated using the polypeptide of SEQ ID NO 221 or 222

SEQ ID NO. 221 (human kappa light chain)

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO. 222 (human lambda light chain)

GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

The DNA sequences of anti-CD 3 molecules such as HC in VH-CH 1-linker-CH 2-CH3 format and LC in VL-CL format are shown in Table 27.

TABLE 25 amino acid sequences of anti-CD 3 antibody arms VH-CH 1-linker-CH 2-CH3 of bispecific antibodies。

TABLE 26 amino acid sequences of anti-CD 3 antibody light chain arms (VL-CL) of bispecific antibodies

TABLE 27 anti-CD 3 arm of bispecific antibody VH-CH 1-linker-CH 2-CH3 format HC and VL-CL format LC cDNA SEQ ID NO。

Engineering BCMA Fab-Fc to generate BCMAxCD3 bispecific

The BCMA specific VH and VL regions were engineered in VH-CH 1-linker-CH 2-CH3 and VL-CL formats, respectively. CD 3-specific VH-CH 1-linker-CH 2-CH3 was generated using a polypeptide comprising Fc silent mutation L234A/L235A/D265S and the CH3 mutation T350V/T366L/K392L/T394W, designed to promote selective heterodimerization, SEQ ID NO 230.

SEQ ID NO:230(huIgG1_G1m(17)_AAS_ZWB)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSREEMTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

BCMA-specific VL-CL is produced using the polypeptide of SEQ ID NO 231 or 232.

SEQ ID NO. 231 (human kappa light chain)

SEQ ID NO. 232 (human lambda light chain)

The amino acid sequences of the BCMA Fab-Fc Heavy (HC) and Light (LC) chains are shown below.

BCMA Fab-Fc heavy chain (SEQ ID NO: 233)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDEGYSSGHYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSREEMTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

BCMA Fab-Fc light chain (SEQ ID NO: 234)

EIVLTQSPGTLSLSPGERATLSCRASQSISSSFLTWYQQKPGQAPRLLIYGASSRATGIPDRFSGGGSGTDFTLTISRLEPEDFAVYYCQHYGSSPMYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Engineering BCMA scFv-Fc to generate BCMAxCD3 bispecific

The BCMA VH/VL region engineered into scFv in either the VH-linker-VL or VL-linker-VH orientation was further engineered to contain an Fc silent mutation (L234A/L235A/D265S) and a T350V/T366L/K392L/T394W mutation designed to promote selective heterodimerization and expressed as an IgG1 (Table 28) using the linker of SEQ ID NO. 3 (Table 2) as described in example 2. The polypeptide of SEQ ID NO. 235 is used as constant domain hinge-CH 2-CH3 (Fc).

SEQ ID NO. 235 (huIgG 1. Sup. G1m (17) -hinge-Fc. Sup. C220S. Sup. AAS ZWB)

EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSREEMTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

TABLE 28 amino acid sequences of anti-BCMA scFv-Fc for the production of BCMA xCD3 bispecific antibodies

The thermal stability, ability to bind T cells, and cytotoxicity of bsAb were determined.

Analysis of thermal stability

For thermal stability, thermal unfolding and aggregation were measured on a nanosampler PromethiusNT.48 instrument using a ramp rate of 1℃per minute from 20℃to 95 ℃. The samples were then transferred by capillary action into nanoDSF capillaries, assayed in parallel (table 2). All variants (except BC3B 126) had similar Tonset, tm1 and Tagg, indicating that specific back mutations and types of mutations at VH N106 have no significant effect on thermostability. bsAb has on average Tonset, tm1 and Tagg at 61 ℃, 68 ℃ and 77 ℃, where Tm1 represents the melting temperature of the scFv moiety, indicating that all variants of Cris-7 can be used to develop therapeutics.

Table thermal stability analysis of bsAb comprising Cris-7 scFv portion。

Activity analysis

BCMAxCD3 bispecific antibodies (BsAb) were tested for their ability to bind T cells or induce T cell-based cytotoxicity against cells expressing each antigen (tables 29 and 30). Binding assays and cytotoxicity assays are described below.

Determination of bsAb binding to T cells and induction of T cell mediated targeting H929 cellsCRL-9068 ^TM ) Is effective in inhibiting cytotoxicity. For T cell binding, bsAb was prepared at a 2x concentration of 600nM in assay medium (RPMI 1640+10% HI-FBS) and then diluted at 3-fold serial dilutions in Greiner sterile polypropylene (PP) plate in staining buffer for 11-point titration. Antibody-free staining buffer was added to the bottom 4 wells and designated as background control (secondary antibody alone). The parents Cris7B, CD3B695 (bivalent mAb control) and CD3B375 (bsAb, which is monovalent for Cris-7B) were also added, as well as the complete dose-response curve. />

Frozen T cells were thawed in a 37℃water bath and gently transferred to 1X 10 vials with 5mL warmed medium (RPMI+10% FBS)/1 vial ⁶ In conical tubes of individual cells. The cells were mixed, centrifuged at 400Xg for 5 min, then resuspended in running staining buffer, counted and checked for viability. Cells were then plated into assay plates at 50 μl/50,000 cells/well. The assay positive control mAb was added in four replicates at 2x and 20nM to 4 wells at the bottom of the first column. The staining buffer without mAb was added to the top 4 wells and designated as background control (secondary antibody alone). Serial dilutions of antibody samples were added at 50 μl/well using an intersa via, incubation at 37 ℃ for 1 hour, according to the attached plate diagram. After 1 hour incubation, 150 μl of staining buffer was added to all wells and the cells were spun at 500xg for 5 minutes to pellet the cells. Cells were washed and then a solution of Alexa Fluor 647 (a 647) conjugated anti-human IgG Fc specific secondary detection antibody at a concentration of 2 μg/mL in staining buffer was added. The secondary detection antibody was added to the washed cells in an amount of 50. Mu.L/well. The plates were covered with foil and incubated on ice or in a refrigerator for 30 minutes. mu.L of staining buffer was added to all wells and the cells were spun at 500Xg for 5 minutes to pellet the cells. Cells were resuspended in 20. Mu.L of SYTOX containing 1:1000 dilution ^TM In running buffer of Green dead cell stain, inThese plates were run on a screen flow cytometer (Essen BioScience, inc.). Briefly, cells were gated on FCS versus SCS scatter plots to eliminate debris. Gating single cells on SCS-A vs SCS-H scatter plot, selecting SYTOX for B1 channel from single cell population ^TM Green viability stain (Thermo Fisher) selectively stained low/negative cells to gate for viable cells. Cell binding of control mAb or test group supernatants was assessed by comparison with negative/isotype control binding generated by RL1 (a 647) geometry from a live cell population.

Antibody sample preparation

BsAb was prepared at 20nM in assay medium, then serially diluted 3-fold in assay medium for 11-point titration and stored at 4 ℃.

The target cell (H929,CRL-9068 ^TM ) By mixing at 5. Mu.L/1X 10 ⁶ Individual cells (50 μl/1 million cells) were prepared by adding the Fc block and then incubated at 37 ℃ for 20 minutes at room temperature. Diluting cells to 4X 10 ⁵ Individual cells/ml were used for plating. Control wells were supplemented with 50 μl of medium per well and incubated at 37 ℃. T cell vials were thawed at 37 ℃ into 50mL conical tubes containing room temperature assay medium (5 mL medium/1 vial of cells). Cells were taken, spun at 300Xg for 5 minutes, resuspended in 10mL fresh medium and counted. T cell suspensions were prepared at a density of 2X 10≡6 cells/mL for E:T ratios of 5:1. According to the plate diagram, cells were prepared in an amount of 50. Mu.L/100 k/well in an assay plate that had been loaded with tumor target cells (from the above step). Control wells were supplemented with 50 μl of medium per well and incubated at 37 ℃.

Antibodies were added at 100 μl/well and serial dilutions were made in assay plates containing mixed tumor target cells and T cells starting at 10 nM. Target cells were 20,000 cells/well and the cell count of pan T cells was 100,000 cells/well. The total assay volume was 200. Mu.L/well. The plates were placed in a humidified cell culture incubator and incubated at 37 ℃ and 5% CO2 for 48 hours.

Cells were washed by adding 150 μl BD staining buffer and then centrifuged at 300xg for 5 min. The staining solution mixture in the BD staining buffer contains:

APC conjugated anti-human CD4 (1:100) (R & D Systems FAB 3791A-025)

APC conjugated anti-human CD8 (1:100) (R & D Systems FAB 1509A-025)

Brilliant Violet 421 ^TM Conjugated anti-human CD25 (1:500)

Preparation of vybrants at 1:12500 dilution separately ^TM DyeCycle ^TM Green stain (Invitrogen) was added to all wells of the assay plate in an amount of 50. Mu.L/Kong Ranse solution mix. Will be thinThe released emerald dye was added to the 2 wells on top of the 1 st control column of all assay plates at 25 μl/well, followed by incubation at room temperature for 20 minutes. Plates were washed 2 times with 150. Mu.L staining buffer and resuspended in 30. Mu.L containing SYTOX ^TM Green Live/read stain (1:1000)In running buffer, use +.>PLUS Screener was analyzed within 4 hours.

Cell-to-cell gating was performed on FSC-H vs SSC-H, and gating was performed on APC (RL 1) vs SSC-H from all cell vs T cell population and tumor cell population. Both tumor cells and T cells were gated on Live and Dead cells (Live/Dead stain) from their respective scatter plots on FSC-H versus Sytox Green (BL 2). Using live T cells, the activated/CD 25 positive T cell population was gated on FSC-H versus Brilliant Violet (VL 1). Cell populations were assayed as follows:

dead tumor cells = SytoxGreen live-dead stain positive cells/total tumor cells x 100

% live T cells = L/D negative T cells/total T cells x 100

% activated T cells = CD25 positive viable T cells/viable T cells x 100

Cell binding analysis showed that the Cris-7 variants showed a range of affinities for T cells, and that this cell-based affinity was correlated with the cytotoxic EC50 (Table 29). Generally, the Cris-7v region variant formatted as scFv in heavy chain-light chain orientation showed about 10 times tighter binding to cells than LH orientation, consistent with ELISA data. The nature of the mutation that eliminates the risk of deamidation based on N106 has a significant impact on T cell binding and cytotoxicity. In summary, three different sets of back mutations (defined by CD3B2030, CD3B2051 and CD3B 2089) in combination with the N106Q/a/G/S mutation resulted in a set of Cris-7 variants with EC50 for T cell binding in the range of 3nM to about 300nM and associated EC50 for cytotoxicity in the range of 0.012nM to 3.5 nM. For T cell redirecting bsAb (bsTCE), a weaker affinity T cell redirection may allow for the design of antibodies to maximize efficacy while minimizing toxicity associated with aberrant T cell activation, accumulation in secondary lymphoid organs, and cytokine release-related toxicity, relative to the affinity of tumor targeting. Furthermore, the "LH" oriented Cris-7 derived scFv bound poorly to T cells compared to HL orientation. Thus, this group was considered advantageous, so we selected a subset of these Cris-7 variants to show the range of binding affinities for the lead bsTCE.

TABLE 29 analysis of T cell binding and cytotoxicity Using H929 cells for Cris-7 XBCMA bsAb。

TABLE 30 functional Activity of bispecific proteins。

EXAMPLE 3 expression and purification of bispecific CD79bxCD3 antibodies and trispecific CD79bxCD20xCD3 antibodies

The CD79bxCD3 bispecific antibody (bsAb) is an immunoglobulin (Ig) G1 bispecific antibody that can bind to Cluster of Differentiation (CD) 3 receptor complex on T lymphocytes and CD79B on B lymphocytes simultaneously or separately. The CD79bxCD20xCD3 trispecific antibody is an immunoglobulin (Ig) G1 trispecific antibody that can bind simultaneously or separately to the CD3 receptor complex on T lymphocytes, the CD20 receptor complex on B lymphocytes and the CD79B receptor complex on B lymphocytes. The antibodies have mutations that reduce Fc binding to fcγ receptors, and heterodimerization has been enhanced by using knob-to-socket structure platform mutations. The trispecific antibodies were developed to evaluate the therapeutic potential of dual targeting CD20 and CD79b for T cell redirection. A schematic of an exemplary CD79bxCD20xCD3 antibody is depicted in fig. 6.

Table 31 provides a summary of examples of some of the CD79b x CD20x CD3 trispecific antibodies described herein.

TABLE 31 exemplary CD79b×CD20×CD3 trispecific antibodies

1463 trispecific Ab CD3-CD20 arm of SEQ ID NO

EIVLTQSPATLSASPGERVTLSCSASSSVSYMNWYQQKPGQAPRRWIYDSSKLASGVPARFSGSGSGRDYTLTISSLEPEDFAVYYCQQWSRNPPTFGGGTKVEIKGGSEGKSSGSGSESKSTGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTRSTMHWVKQAPGQGLEWIGYINPSSAYTNYNQKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCASPQVHYDYAGFPYWGQGTLVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGSEGKSSGSGSESKSTGGSAIQLTQSPSSLSASVGDRVTITCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWTSNPPTFGQGTKLEIKGGSEGKSSGSGSESKSTGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGAIYPGNGDTSYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSS

SEQ ID NO:1464 trispecific Ab CD3-CD20 arm

GAGATCGTGCTGACCCAGTCTCCTGCCACACTGAGTGCTTCTCCAGGCGAGAGAGTGACCCTGTCCTGCTCCGCTTCCTCCTCCGTGTCCTACATGAACTGGTATCAGCAGAAGCCCGGCCAGGCTCCTCGGAGATGGATCTACGACTCTTCCAAGCTGGCCTCTGGTGTGCCAGCCAGATTTTCTGGCTCTGGCTCCGGCAGAGACTATACCCTGACCATCTCCAGCCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTGGTCTAGGAACCCTCCTACCTTTGGCGGAGGCACCAAGGTGGAAATCAAGGGCGGATCTGAGGGAAAGTCCAGCGGCTCCGGCAGCGAAAGCAAGTCCACCGGCGGAAGCCAGGTTCAACTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCTCCTCCGTCAAGGTGTCCTGCAAGGCTTCCGGCTACACCTTTACCAGATCCACCATGCACTGGGTCAAGCAGGCCCCTGGACAAGGCTTGGAGTGGATCGGCTACATCAACCCCAGCTCCGCCTACACCAACTACAACCAGAAATTCCAGGGCAGAGTGACCCTGACCGCCGACAAGTCTACCTCCACCGCCTACATGGAACTGTCCAGCCTGAGATCTGAGGACACCGCCGTGTACTACTGCGCCTCTCCTCAGGTCCACTACGACTACGCCGGCTTTCCTTATTGGGGCCAGGGCACACTGGTCACCGTTTCTTCTGAGCCCAAATCTAGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGAGCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTGTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGTCTCTCTCCCTGTCTCCGGGAAAAGGAGGGAGCGAGGGAAAGTCCAGCGGAAGCGGCTCTGAGTCCAAATCCACCGGAGGGAGCGCCATTCAGCTGACCCAGTCTCCATCCTCTCTGTCCGCCTCTGTGGGCGACAGAGTGACAATTACCTGCCGGGCCTCCTCCTCCGTGTCCTACATCCATTGGTTCCAGCAGAAGCCCGGCAAGGCCCCTAAGCCTCTGATCTACGCCACCTCCAATCTGGCCTCTGGCGTGCCCTCCAGATTTTCCGGATCTGGCTCTGGAACCGACTTTACCCTGACAATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGTCAGCAGTGGACCAGCAATCCTCCTACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGGGCGGCTCCGAGGGCAAGAGCAGCGGCAGCGGCAGCGAGAGCAAGAGCACCGGCGGCAGCCAGGTTCAGCTGGTTCAGTCTGGTGCCGAAGTGAAGAAACCTGGCTCCTCCGTGAAAGTGTCCTGCAAGGCTTCCGGCTACACTTTTACCAGCTACAACATGCACTGGGTCCGACAGGCCCCTGGACAAGGATTGGAATGGATGGGCGCTATCTACCCCGGCAACGGCGATACCTCTTACGCCCAGAAATTCCAGGGCAGAGTGACCATCACCGCCGACAAGTCTACCTCCACCGCCTACATGGAACTGTCCAGCCTGAGATCTGAGGACACCGCCGTGTACTACTGCGCCCGGTCTACCTATTATGGCGGCGACTGGTACTTCAACGTGTGGGGCCAGGGAACCCTGGTCACAGTCTCTTCT

1465 trispecific Ab CD3-CD20 arm of SEQ ID NO

EIVLTQSPATLSASPGERVTLSCSASSSVSYMNWYQQKPGQAPRRWIYDSSKLASGVPARFSGSGSGRDYTLTISSLEPEDFAVYYCQQWSRNPPTFGGGTKVEIKGGSEGKSSGSGSESKSTGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTRSTMHWVRQAPGQGLEWMGYINPSSAYTNYAQKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCASPQVHYDYGGFPYWGQGTLVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGSEGKSSGSGSESKSTGGSAIQLTQSPSSLSASVGDRVTITCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWTSNPPTFGQGTKLEIKGGSEGKSSGSGSESKSTGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGAIYPGNGDTSYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSS

1466 trispecific Ab CD3-CD20 arm of SEQ ID NO

GAGATCGTGCTGACCCAGTCTCCTGCCACACTGAGTGCTTCTCCAGGCGAGAGAGTGACCCTGTCCTGCTCCGCTTCCTCCTCCGTGTCCTACATGAACTGGTATCAGCAGAAGCCCGGCCAGGCTCCTCGGAGATGGATCTACGACTCTTCCAAGCTGGCCTCTGGTGTGCCAGCCAGATTTTCTGGCTCTGGCTCCGGCAGAGACTATACCCTGACCATCTCCAGCCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTGGTCTAGGAACCCTCCTACCTTTGGCGGAGGCACCAAGGTGGAAATCAAGGGCGGATCTGAGGGAAAGTCCAGCGGCTCCGGCAGCGAAAGCAAGTCCACCGGCGGAAGCCAGGTTCAACTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCTCCTCCGTGAAAGTGTCCTGCAAGGCTTCCGGCTACACTTTTACCAGATCCACCATGCACTGGGTCCGACAGGCTCCAGGACAAGGCTTGGAGTGGATGGGCTACATCAACCCCAGCTCCGCCTACACCAACTACGCCCAGAAATTCCAGGGCAGAGTGACCCTGACCGCCGACAAGTCTACCTCCACCGCCTACATGGAACTGTCCAGCCTGAGATCTGAGGACACCGCCGTGTACTACTGCGCTTCTCCTCAGGTGCACTACGACTACGGCGGCTTTCCTTATTGGGGCCAGGGCACACTGGTCACCGTTTCTTCTGAGCCCAAATCTAGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGAGCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTGTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGTCTCTCTCCCTGTCTCCGGGAAAAGGAGGGAGCGAGGGAAAGTCCAGCGGAAGCGGCTCTGAGTCCAAATCCACCGGAGGGAGCGCCATTCAGCTGACCCAGTCTCCATCCTCTCTGTCCGCCTCTGTGGGCGACAGAGTGACAATTACCTGCCGGGCCTCCTCCTCCGTGTCCTACATCCATTGGTTCCAGCAGAAGCCCGGCAAGGCCCCTAAGCCTCTGATCTACGCCACCTCCAATCTGGCCTCTGGCGTGCCCTCCAGATTTTCCGGATCTGGCTCTGGAACCGACTTTACCCTGACAATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGTCAGCAGTGGACCAGCAATCCTCCTACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGGGCGGCTCCGAGGGCAAGAGCAGCGGCAGCGGCAGCGAGAGCAAGAGCACCGGCGGCAGCCAGGTTCAGCTGGTTCAGTCTGGTGCCGAAGTGAAGAAACCTGGCTCCTCCGTGAAAGTGTCCTGCAAGGCTTCCGGCTACACTTTTACCAGCTACAACATGCACTGGGTCCGACAGGCCCCTGGACAAGGATTGGAATGGATGGGCGCTATCTACCCCGGCAACGGCGATACCTCTTACGCCCAGAAATTCCAGGGCAGAGTGACCATCACCGCCGACAAGTCTACCTCCACCGCCTACATGGAACTGTCCAGCCTGAGATCTGAGGACACCGCCGTGTACTACTGCGCCCGGTCTACCTATTATGGCGGCGACTGGTACTTCAACGTGTGGGGCCAGGGAACCCTGGTCACAGTCTCTTCT

1467 trispecific Ab CD3-CD20 arm of SEQ ID NO

DIQMTQSPSSLSASVGDRVTITCRARQSIGTAIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSGSWPYTFGQGTKLEIKGGSEGKSSGSGSESKSTGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSRYNMNWVRQAPGKGLEWVSSISTSSNYIYYADSVKGRFTFSRDNAKNSLDLQMSGLRAEDTAIYYCTRGWGPFDYWGQGTLVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWTSNPPTFGQGTKLEIKGGSEGKSSGSGSESKSTGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGAIYPGNGDTSYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSS

1468 trispecific Ab CD3-CD20 arm of SEQ ID NO

GACATACAAATGACACAATCACCCTCTTCTCTTTCTGCAAGCGTTGGCGACCGTGTCACTATCACTTGTCGAGCCCGCCAGTCCATAGGTACTGCCATTCACTGGTATCAACAGAAGCCTGGCAAGGCTCCCAAACTCCTGATTAAGTATGCCAGCGAGAGCATTTCCGGCGTACCTTCAAGATTTTCCGGCTCCGGTAGTGGGACAGATTTCACTCTCACTATATCTAGCCTCCAACCAGAAGATTTCGCCACTTACTACTGTCAACAATCAGGTTCATGGCCTTACACTTTCGGCCAGGGGACAAAATTGGAGATCAAGGGCGGCTCCGAGGGCAAGAGCAGCGGCAGCGGCAGCGAGAGCAAGAGCACCGGCGGCAGCGAGGTGCAACTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGATATAACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTACTAGTAGTAATTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCTTCTCCAGAGACAACGCCAAGAACTCACTGGATCTGCAAATGAGCGGCCTGAGAGCCGAGGACACGGCTATTTATTACTGTACGAGAGGCTGGGGGCCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGAGCCCAAATCTAGCGACAAAACTCACACATGTCCACCGTGCCCAGCACCTGAAGCAGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGAGCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGTCTAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGAGGCGGAGGGAGTGGCGGGGGAGGCTCTGCAATCCAAC TAACTCAAAGTCCAAGTAGTCTGTCTGCTTCCGTGGGCGACAGAGTGACAATCACCTGTAGAGCCTCCAGCAGCGTCTCCTACATCCACTGGTTCCAGCAAAAACCTGGCAAGGCCCCTAAGCCTCTGATCTACGCCACCTCCAACCTGGCCTCTGGCGTGCCCTCTCGGTTCTCCGGCTCTGGCTCCGGAACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGGATTTTGCTACCTACTACTGCCAGCAGTGGACCTCTAACCCTCCAACATTCGGCCAGGGCACCAAGCTGGAAATCAAGGGCGGCTCCGAGGGCAAGAGCAGCGGCAGCGGCAGCGAGAGCAAGAGCACCGGCGGCAGCCAAGTGCAATTAGTGCAAAGTGGTGCAGAAGTCAAGAAGCCTGGAAGCTCCGTGAAAGTGTCCTGCAAGGCCTCTGGCTACACCTTTACCTCCTACAACATGCACTGGGTGCGGCAGGCTCCTGGCCAGGGCCTGGAGTGGATGGGCGCTATCTACCCCGGCAACGGCGATACCTCTTACGCCCAGAAGTTCCAGGGCAGAGTGACCATCACCGCCGACAAGTCCACATCTACAGCCTACATGGAACTGTCCTCCCTGCGGTCCGAGGACACCGCTGTGTACTATTGTGCCAGATCTACCTACTACGGCGGCGACTGGTACTTCAACGTGTGGGGCCAAGGAACCCTGGTGACCGTGTCTAGC

1469 trispecific Ab CD3-CD20 arm of SEQ ID NO

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTRSTMHWVRQAPGQGLEWMGYINPSSAYTNYAQKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCASPQVHYDYGGFPYWGQGTLVTVSSGGSEGKSSGSGSESKSTGGSEIVLTQSPATLSASPGERVTLSCSASSSVSYMNWYQQKPGQAPRRWIYDSSKLASGVPARFSGSGSGRDYTLTISSLEPEDFAVYYCQQWSRNPPTFGGGTKVEIKEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWTSNPPTFGQGTKLEIKGGSEGKSSGSGSESKSTGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGAIYPGNGDTSYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSS

1470 trispecific Ab CD3-CD20 arm of SEQ ID NO

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCTGGCAGCAGCGTGAAGGTGAGCTGTAAGGCCAGCGGCTACACTTTCACTAGGAGCACTATGCACTGGGTGAGGCAGGCCCCTGGCCAGGGCCTGGAGTGGATGGGCTACATCAATCCTAGCAGCGCCTACACTAATTACGCCCAGAAGTTCCAGGGCAGGGTGACTCTGACTGCCGATAAGAGCACTAGCACTGCCTACATGGAGCTGAGCAGCCTGAGGAGCGAGGATACTGCCGTGTACTACTGTGCCAGCCCTCAGGTGCACTACGATTACGGCGGCTTCCCTTACTGGGGCCAGGGCACTCTGGTGACTGTGAGCAGCGGCGGCTCCGAGGGCAAGAGCAGCGGCAGCGGCAGCGAGAGCAAGAGCACCGGCGGCAGCGAGATCGTGCTGACTCAGAGCCCTGCCACTCTGAGCGCCAGCCCTGGCGAGAGGGTGACTCTGAGCTGTAGCGCCAGCAGCAGCGTGAGCTACATGAATTGGTACCAGCAGAAGCCTGGCCAGGCCCCTAGGAGGTGGATCTACGATAGCAGCAAGCTGGCCAGCGGCGTGCCTGCCAGGTTCAGCGGCAGCGGCAGCGGCAGGGATTACACTCTGACTATCAGCAGCCTGGAGCCTGAGGATTTCGCCGTGTACTACTGTCAGCAGTGGAGCAGGAATCCTCCTACTTTCGGCGGCGGCACTAAGGTGGAGATCAAGGAGCCCAAATCTAGCGACAAAACTCACACATGTCCACCGTGCCCAGCACCTGAAGCAGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGAGCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGTCTAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGAGGCGGAGGGAGTGGCGGGGGAGGCTCTGCAATCCAACTAACTCAAAGTCCAAGTAGTCTGTCTGCTTCCGTGGGCGACAGAGTGACAATCACCTGTAGAGCCTCCAGCAGCGTCTCCTACATCCACTGGTTCCAGCAAAAACCTGGCAAGGCCCCTAAGCCTCTGATCTACGCCACCTCCAACCTGGCCTCTGGCGTGCCCTCTCGGTTCTCCGGCTCTGGCTCCGGAACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGGATTTTGCTACCTACTACTGCCAGCAGTGGACCTCTAACCCTCCAACATTCGGCCAGGGCACCAAGCTGGAAATCAAGGGCGGCTCCGAGGGCAAGAGCAGCGGCAGCGGCAGCGAGAGCAAGAGCACCGGCGGCAGCCAAGTGCAATTAGTGCAAAGTGGTGCAGAAGTCAAGAAGCCTGGAAGCTCCGTGAAAGTGTCCTGCAAGGCCTCTGGCTACACCTTTACCTCCTACAACATGCACTGGGTGCGGCAGGCTCCTGGCCAGGGCCTGGAGTGGATGGGCGCTATCTACCCCGGCAACGGCGATACCTCTTACGCCCAGAAGTTCCAGGGCAGAGTGACCATCACCGCCGACAAGTCCACATCTACAGCCTACATGGAACTGTCCTCCCTGCGGTCCGAGGACACCGCTGTGTACTATTGTGCCAGATCTACCTACTACGGCGGCGACTGGTACTTCAACGTGTGGGGCCAAGGAACCCTGGTGACCGTGTCTAGC

1471 trispecific Ab CD3-CD20 arm of SEQ ID NO

DIQMTQSPSSLSASVGDRVTITCRARQSIGTAIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSGSWPYTFGQGTKLEIKGGSEGKSSGSGSESKSTGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSRYNMNWVRQAPGKGLEWVSSISTSSNYIYYADSVKGRFTFSRDNAKNSLDLQMSGLRAEDTAIYYCTRGWGPFDYWGQGTLVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPQVWIYATSNLASGV PVRFSGSGSGTSYSLTISRVEAEDTATYYCQQWIFNPPTFGSGTKLEIRGGSEGKSSGSGSESKSTGGSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVYYGSNYWYFDVWGTGTTVTVSS

1472 trispecific Ab CD3-CD20 arm of SEQ ID NO

GACATACAAATGACACAATCACCCTCTTCTCTTTCTGCAAGCGTTGGCGACCGTGTCACTATCACTTGTCGAGCCCGCCAGTCCATAGGTACTGCCATTCACTGGTATCAACAGAAGCCTGGCAAGGCTCCCAAACTCCTGATTAAGTATGCCAGCGAGAGCATTTCCGGCGTACCTTCAAGATTTTCCGGCTCCGGTAGTGGGACAGATTTCACTCTCACTATATCTAGCCTCCAACCAGAAGATTTCGCCACTTACTACTGTCAACAATCAGGTTCATGGCCTTACACTTTCGGCCAGGGGACAAAATTGGAGATCAAGGGCGGCTCCGAGGGCAAGAGCAGCGGCAGCGGCAGCGAGAGCAAGAGCACCGGCGGCAGCGAGGTGCAACTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGATATAACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTACTAGTAGTAATTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCTTCTCCAGAGACAACGCCAAGAACTCACTGGATCTGCAAATGAGCGGCCTGAGAGCCGAGGACACGGCTATTTATTACTGTACGAGAGGCTGGGGGCCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGAGCCCAAATCTAGCGACAAAACTCACACATGTCCACCGTGCCCAGCACCTGAAGCAGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGAGCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG AGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGTCTAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGAGGCGGAGGGAGTGGCGGGGGAGGCTCTCAAATAGTCCTTTCACAGTCCCCAGCTATTCTTTCAGCCTCTCCCGGTGAAAAGGTTACAATGACCTGCCGGGCAAGCTCCAGTGTCTCATATATGCACTGGTACCAACAAAAACCTGGCAGTAGTCCTCAGGTGTGGATCTACGCTACAAGCAATCTCGCTTCCGGGGTTCCCGTGAGGTTTAGCGGAAGCGGGTCTGGAACTAGTTATTCCTTGACAATTAGTCGGGTTGAAGCCGAGGACACCGCCACTTACTATTGCCAACAGTGGATATTCAATCCACCCACCTTCGGTTCAGGTACCAAGCTCGAAATCCGTGGCGGCTCCGAGGGCAAGAGCAGCGGCAGCGGCAGCGAGAGCAAGAGCACCGGCGGCAGCCAAGCATATCTGCAACAGAGCGGAGCTGAGCTGGTTCGGCCTGGCGCCTCTGTAAAAATGAGTTGCAAGGCCAGTGGTTATACATTCACATCATATAATATGCACTGGGTAAAGCAAACTCCCCGACAGGGGCTTGAATGGATTGGCGCAATCTATCCCGGCAATGGGGATACATCCTACAATCAGAAATTCAAGGGCAAGGCAACACTGACCGTTGACAAATCCTCATCAACAGCCTACATGCAGCTCAGTTCCCTCACTAGCGAAGATTCTGCTGTGTATTTCTGTGCAAGGGTGTATTATGGTTCTAATTACTGGTATTTCGATGTTTGGGGAACCGGAACTACCGTAACTGTTTCTAGC

1473 trispecific Ab CD3-CD20 arm of SEQ ID NO

DIQMTQSPSSLSASVGDRVTITCRARQSIGTAIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSGSWPYTFGQGTKLEIKGGSEGKSSGSGSESKSTGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSRYNMNWVRQAPGKGLEWVSSISTSSNYIYYADSVKGRFTFSRDNAKNSLDLQMSGLRAEDTAIYYCTRGWGPFDYWGQGTLVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSQIVLSQSPAILSASPGEKVTMTCRASLSVSSMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWIFNPPTFGGGTKLEIKGGSEGKSSGSGSESKSTGGSQAYLQQSGAELVRPGASVKMSCKTSGYTFSSYNMHWVKQTPRQALEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCTRSNYYGSSGWYFDVWGTGTTVTVSS

1474 trispecific Ab CD3-CD20 arm of SEQ ID NO

GACATACAAATGACACAATCACCCTCTTCTCTTTCTGCAAGCGTTGGCGACCGTGTCACTATCACTTGTCGAGCCCGCCAGTCCATAGGTACTGCCATTCACTGGTATCAACAGAAGCCTGGCAAGGCTCCCAAACTCCTGATTAAGTATGCCAGCGAGAGCATTTCCGGCGTACCTTCAAGATTTTCCGGCTCCGGTAGTGGGACAGATTTCACTCTCACTATATCTAGCCTCCAACCAGAAGATTTCGCCACTTACTACTGTCAACAATCAGGTTCATGGCCTTACACTTTCGGCCAGGGGACAAAATTGGAGATCAAGGGCGGCTCCGAGGGCAAGAGCAGCGGCAGCGGCAGCGAGAGCAAGAGCACCGGCGGCAGCGAGGTGCAACTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGATATAACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTACTAGTAGTAATTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCTTCTCCAGAGACAACGCCAAGAACTCACTGGATCTGCAAATGAGCGGCCTGAGAGCCGAGGACACGGCTATTTATTACTGTACGAGAGGCTGGGGGCCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGAGCCCAAATCTAGCGACAAAACTCACACATGTCCACCGTGCCCAGCACCTGAAGCAGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGAGCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGTCTAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGAGGCGGAGGGAGTGGCGGGGGAGGCTCTCAGATTGTCCTGAGCCAATCCCCAGCAATTCTGAGTGCTAGCCCTGGAGAGAAGGTAACAATGACTTGTCGGGCATCCCTTAGCGTCTCATCCATGCATTGGTATCAACAAAAGCCAGGTTCATCTCCAAAACCCTGGATTTACGCTACATCTAACCTGGCATCTGGGGTGCCTGCCAGATTTAGTGGATCTGGTTCCGGCACATCATATTCCCTTACAATCAGCCGAGTGGAAGCCGAGGATGCTGCAACCTATTACTGTCAACAATGGATATTTAACCCTCCCACCTTTGGGGGTGGGACTAAACTCGAAATCAAGGGCGGCTCCGAGGGCAAGAGCAGCGGCAGCGGCAGCGAGAGCAAGAGCACCGGCGGCAGCCAAGCCTATCTTCAACAATCTGGGGCTGAGCTTGTCCGGCCAGGAGCCTCCGTCAAAATGAGCTGCAAAACCTCAGGTTATACTTTTAGTAGCTATAACATGCATTGGGTAAAACAAACCCCCCGACAAGCATTGGAGTGGATAGGGGCCATATACCCCGGCAATGGAGACACAAGTTACAACCAGAAGTTTAAAGGCAAAGCTACACTCACAGTTGACAAATCCTCAAGTACTGCTTATATGCAACTCTCCTCTCTCACTTCCGAAGACAGTGCCGTATATTTTTGCACTCGGTCCAATTACTATGGATCTAGTGGCTGGTACTTTGACGTTTGGGGCACTGGGACAACTGTTACAGTGTCCAGC

1475 trispecific Ab CD3-CD20 arm of SEQ ID NO

EIVLTQSPATLSASPGERVTLSCSASSSVSYMNWYQQKPGQAPRRWIYDSSKLASGVPARFSGSGSGRDYTLTISSLEPEDFAVYYCQQWSRNPPTFGGGTKVEIKGGSEGKSSGSGSESKSTGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTRSTMHWVKQAPGQGLEWIGYINPSSAYTNYNQKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCASPQVHYDYAGFPYWGQGTLVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPQVWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDTATYYCQQWIFNPPTFGSGTKLEIRGGSEGKSSGSGSESKSTGGSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVYYGSNYWYFDVWGTGTTVTVSS

1476 trispecific Ab CD3-CD20 arm of SEQ ID NO

GAGATCGTGCTGACTCAGAGCCCTGCCACTCTGAGCGCCAGCCCTGGCGAGAGGGTGACTCTGAGCTGTAGCGCCAGCAGCAGCGTGAGCTACATGAATTGGTACCAGCAGAAGCCTGGCCAGGCCCCTAGGAGGTGGATCTACGATAGCAGCAAGCTGGCCAGCGGCGTGCCTGCCAGGTTCAGCGGCAGCGGCAGCGGCAGGGATTACACTCTGACTATCAGCAGCCTGGAGCCTGAGGATTTCGCCGTGTACTACTGTCAGCAGTGGAGCAGGAATCCTCCTACTTTCGGCGGCGGCACTAAGGTGGAGATCAAGGGCGGCTCCGAGGGCAAGAGCAGCGGCAGCGGCAGCGAGAGCAAGAGCACCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCTGGCAGCAGCGTGAAGGTGAGCTGTAAGGCCAGCGGCTACACTTTCACTAGGAGCACTATGCACTGGGTGAAGCAGGCCCCTGGCCAGGGCCTGGAGTGGATCGGCTACATCAATCCTAGCAGCGCCTACACTAATTACAATCAGAAGTTCCAGGGCAGGGTGACTCTGACTGCCGATAAGAGCACTAGCACTGCCTACATGGAGCTGAGCAGCCTGAGGAGCGAGGATACTGCCGTGTACTACTGTGCCAGCCCTCAGGTGCACTACGATTACGCCGGCTTCCCTTACTGGGGCCAGGGCACTCTGGTGACTGTGAGCAGCGAGCCCAAATCTAGCGACAAAACTCACACATGTCCACCGTGCCCAGCACCTGAAGCAGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGAGCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGTCTAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGAGGCGGAGGGAGTGGCGGGGGAGGCTCTCAAATAGTCCTTTCACAGTCCCCAGCTATTCTTTCAGCCTCTCCCGGTGAAAAGGTTACAATGACCTGCCGGGCAAGCTCCAGTGTCTCATATATGCACTGGTACCAACAAAAACCTGGCAGTAGTCCTCAGGTGTGGATCTACGCTACAAGCAATCTCGCTTCCGGGGTTCCCGTGAGGTTTAGCGGAAGCGGGTCTGGAACTAGTTATTCCTTGACAATTAGTCGGGTTGAAGCCGAGGACACCGCCACTTACTATTGCCAACAGTGGATATTCAATCCACCCACCTTCGGTTCAGGTACCAAGCTCGAAATCCGTGGCGGCTCCGAGGGCAAGAGCAGCGGCAGCGGCAGCGAGAGCAAGAGCACCGGCGGCAGCCAAGCATATCTGCAACAGAGCGGAGCTGAGCTGGTTCGGCCTGGCGCCTCTGTAAAAATGAGTTGCAAGGCCAGTGGTTATACATTCACATCATATAATATGCACTGGGTAAAGCAAACTCCCCGACAGGGGCTTGAATGGATTGGCGCAATCTATCCCGGCAATGGGGATACATCCTACAATCAGAAATTCAAGGGCAAGGCAACACTGACCGTTGACAAATCCTCATCAACAGCCTACATGCAGCTCAGTTCCCTCACTAGCGAAGATTCTGCTGTGTATTTCTGTGCAAGGGTGTATTATGGTTCTAATTACTGGTATTTCGATGTTTGGGGAACCGGAACTACCGTAACTGTTTCTAGC

1477 trispecific Ab CD3-CD20 arm of SEQ ID NO

EIVLTQSPATLSASPGERVTLSCSASSSVSYMNWYQQKPGQAPRRWIYDSSKLASGVPARFSGSGSGRDYTLTISSLEPEDFAVYYCQQWSRNPPTFGGGTKVEIKGGSEGKSSGSGSESKSTGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTRSTMHWVKQAPGQGLEWIGYINPSSAYTNYNQKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCASPQVHYDYAGFPYWGQGTLVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSQIVLSQSPAILSASPGEKVTMTCRASLSVSSMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWIFNPPTFGGGTKLEIKGGSEGKSSGSGSESKSTGGSQAYLQQSGAELVRPGASVKMSCKTSGYTFSSYNMHWVKQTPRQALEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCTRSNYYGSSGWYFDVWGTGTTVTVSS

1478 trispecific Ab CD3-CD20 arm of SEQ ID NO

GAGATCGTGCTGACTCAGAGCCCTGCCACTCTGAGCGCCAGCCCTGGCGAGAGGGTGACTCTGAGCTGTAGCGCCAGCAGCAGCGTGAGCTACATGAATTGGTACCAGCAGAAGCCTGGCCAGGCCCCTAGGAGGTGGATCTACGATAGCAGCAAGCTGGCCAGCGGCGTGCCTGCCAGGTTCAGCGGCAGCGGCAGCGGCAGGGATTACACTCTGACTATCAGCAGCCTGGAGCCTGAGGATTTCGCCGTGTACTACTGTCAGCAGTGGAGCAGGAATCCTCCTACTTTCGGCGGCGGCACTAAGGTGGAGATCAAGGGCGGCTCCGAGGGCAAGAGCAGCGGCAGCGGCAGCGAGAGCAAGAGCACCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCTGGCAGCAGCGTGAAGGTGAGCTGTAAGGCCAGCGGCTACACTTTCACTAGGAGCACTATGCACTGGGTGAAGCAGGCCCCTGGCCAGGGCCTGGAGTGGATCGGCTACATCAATCCTAGCAGCGCCTACACTAATTACAATCAGAAGTTCCAGGGCAGGGTGACTCTGACTGCCGATAAGAGCACTAGCACTGCCTACATGGAGCTGAGCAGCCTGAGGAGCGAGGATACTGCCGTGTACTACTGTGCCAGCCCTCAGGTGCACTACGATTACGCCGGCTTCCCTTACTGGGGCCAGGGCACTCTGGTGACTGTGAGCAGCGAGCCCAAATCTAGCGACAAAACTCACACATGTCCACCGTGCCCAGCACCTGAAGCAGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGAGCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGTCTAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGAGGCGGAGGGAGTGGCGGGGGAGGCTCTCAGATTGTCCTGAGCCAATCCCCAGCAATTCTGAGTGCTAGCCCTGGAGAGAAGGTAACAATGACTTGTCGGGCATCCCTTAGCGTCTCATCCATGCATTGGTATCAACAAAAGCCAGGTTCATCTCCAAAACCCTGGATTTACGCTACATCTAACCTGGCATCTGGGGTGCCTGCCAGATTTAGTGGATCTGGTTCCGGCACATCATATTCCCTTACAATCAGCCGAGTGGAAGCCGAGGATGCTGCAACCTATTACTGTCAACAATGGATATTTAACCCTCCCACCTTTGGGGGTGGGACTAAACTCGAAATCAAGGGCGGCTCCGAGGGCAAGAGCAGCGGCAGCGGCAGCGAGAGCAAGAGCACCGGCGGCAGCCAAGCCTATCTTCAACAATCTGGGGCTGAGCTTGTCCGGCCAGGAGCCTCCGTCAAAATGAGCTGCAAAACCTCAGGTTATACTTTTAGTAGCTATAACATGCATTGGGTAAAACAAACCCCCCGACAAGCATTGGAGTGGATAGGGGCCATATACCCCGGCAATGGAGACACAAGTTACAACCAGAAGTTTAAAGGCAAAGCTACACTCACAGTTGACAAATCCTCAAGTACTGCTTATATGCAACTCTCCTCTCTCACTTCCGAAGACAGTGCCGTATATTTTTGCACTCGGTCCAATTACTATGGATCTAGTGGCTGGTACTTTGACGTTTGGGGCACTGGGACAACTGTTACAGTGTCCAGC

1479 trispecific Ab CD3-CD20 arm of SEQ ID NO

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTRSTMHWVRQAPGQGLEWMGYINPSSAYTNYAQKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCASPQVHYDYGGFPYWGQGTLVTVSSGGSEGKSSGSGSESKSTGGSEIVLTQSPATLSASPGERVTLSCSASSSVSYMNWYQQKPGQAPRRWIYDSSKLASGVPARFSGSGSGRDYTLTISSLEPEDFAVYYCQQWSRNPPTFGGGTKVEIKEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPQVWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDTATYYCQQWIFNPPTFGSGTKLEIRGGSEGKSSGSGSESKSTGGSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVYYGSNYWYFDVWGTGTTVTVSSSEQ ID NO 1480 III Specific Ab CD3-CD20 arm

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCTGGCAGCAGCGTGAAGGTGAGCTGTAAGGCCAGCGGCTACACTTTCACTAGGAGCACTATGCACTGGGTGAGGCAGGCCCCTGGCCAGGGCCTGGAGTGGATGGGCTACATCAATCCTAGCAGCGCCTACACTAATTACGCCCAGAAGTTCCAGGGCAGGGTGACTCTGACTGCCGATAAGAGCACTAGCACTGCCTACATGGAGCTGAGCAGCCTGAGGAGCGAGGATACTGCCGTGTACTACTGTGCCAGCCCTCAGGTGCACTACGATTACGGCGGCTTCCCTTACTGGGGCCAGGGCACTCTGGTGACTGTGAGCAGCGGCGGCTCCGAGGGCAAGAGCAGCGGCAGCGGCAGCGAGAGCAAGAGCACCGGCGGCAGCGAGATCGTGCTGACTCAGAGCCCTGCCACTCTGAGCGCCAGCCCTGGCGAGAGGGTGACTCTGAGCTGTAGCGCCAGCAGCAGCGTGAGCTACATGAATTGGTACCAGCAGAAGCCTGGCCAGGCCCCTAGGAGGTGGATCTACGATAGCAGCAAGCTGGCCAGCGGCGTGCCTGCCAGGTTCAGCGGCAGCGGCAGCGGCAGGGATTACACTCTGACTATCAGCAGCCTGGAGCCTGAGGATTTCGCCGTGTACTACTGTCAGCAGTGGAGCAGGAATCCTCCTACTTTCGGCGGCGGCACTAAGGTGGAGATCAAGGAGCCCAAATCTAGCGACAAAACTCACACATGTCCACCGTGCCCAGCACCTGAAGCAGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGAGCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGTCTAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGAGGCGGAGGGAGTGGCGGGGGAGGCTCTCAAATAGTCCTTTCACAGTCCCCAGCTATTCTTTCAGCCTCTCCCGGTGAAAAGGTTACAATGACCTGCCGGGCAAGCTCCAGTGTCTCATATATGCACTGGTACCAACAAAAACCTGGCAGTAGTCCTCAGGTGTGGATCTACGCTACAAGCAATCTCGCTTCCGGGGTTCCCGTGAGGTTTAGCGGAAGCGGGTCTGGAACTAGTTATTCCTTGACAATTAGTCGGGTTGAAGCCGAGGACACCGCCACTTACTATTGCCAACAGTGGATATTCAATCCACCCACCTTCGGTTCAGGTACCAAGCTCGAAATCCGTGGCGGCTCCGAGGGCAAGAGCAGCGGCAGCGGCAGCGAGAGCAAGAGCACCGGCGGCAGCCAAGCATATCTGCAACAGAGCGGAGCTGAGCTGGTTCGGCCTGGCGCCTCTGTAAAAATGAGTTGCAAGGCCAGTGGTTATACATTCACATCATATAATATGCACTGGGTAAAGCAAACTCCCCGACAGGGGCTTGAATGGATTGGCGCAATCTATCCCGGCAATGGGGATACATCCTACAATCAGAAATTCAAGGGCAAGGCAACACTGACCGTTGACAAATCCTCATCAACAGCCTACATGCAGCTCAGTTCCCTCACTAGCGAAGATTCTGCTGTGTATTTCTGTGCAAGGGTGTATTATGGTTCTAATTACTGGTATTTCGATGTTTGGGGAACCGGAACTACCGTAACTGTTTCTAGC

1481 trispecific Ab CD3-CD20 arm of SEQ ID NO

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTRSTMHWVRQAPGQGLEWMGYINPSSAYTNYAQKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCASPQVHYDYGGFPYWGQGTLVTVSSGGSEGKSSGSGSESKSTGGSEIVLTQSPATLSASPGERVTLSCSASSSVSYMNWYQQKPGQAPRRWIYDSSKLASGVPARFSGSGSGRDYTLTISSLEPEDFAVYYCQQWSRNPPTFGGGTKVEIKEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSQIVLSQSPAILSASPGEKVTMTCRASLSVSSMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWIFNPPTFGGGTKLEIKGGSEGKSSGSGSESKSTGGSQAYLQQSGAELVRPGASVKMSCKTSGYTFSSYNMHWVKQTPRQALEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCTRSNYYGSSGWYFDVWGTGTTVTVSSSEQ ID NO 1482 three Specific Ab CD3-CD20 arm

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCTGGCAGCAGCGTGAAGGTGAGCTGTAAGGCCAGCGGCTACACTTTCACTAGGAGCACTATGCACTGGGTGAGGCAGGCCCCTGGCCAGGGCCTGGAGTGGATGGGCTACATCAATCCTAGCAGCGCCTACACTAATTACGCCCAGAAGTTCCAGGGCAGGGTGACTCTGACTGCCGATAAGAGCACTAGCACTGCCTACATGGAGCTGAGCAGCCTGAGGAGCGAGGATACTGCCGTGTACTACTGTGCCAGCCCTCAGGTGCACTACGATTACGGCGGCTTCCCTTACTGGGGCCAGGGCACTCTGGTGACTGTGAGCAGCGGCGGCTCCGAGGGCAAGAGCAGCGGCAGCGGCAGCGAGAGCAAGAGCACCGGCGGCAGCGAGATCGTGCTGACTCAGAGCCCTGCCACTCTGAGCGCCAGCCCTGGCGAGAGGGTGACTCTGAGCTGTAGCGCCAGCAGCAGCGTGAGCTACATGAATTGGTACCAGCAGAAGCCTGGCCAGGCCCCTAGGAGGTGGATCTACGATAGCAGCAAGCTGGCCAGCGGCGTGCCTGCCAGGTTCAGCGGCAGCGGCAGCGGCAGGGATTACACTCTGACTATCAGCAGCCTGGAGCCTGAGGATTTCGCCGTGTACTACTGTCAGCAGTGGAGCAGGAATCCTCCTACTTTCGGCGGCGGCACTAAGGTGGAGATCAAGGAGCCCAAATCTAGCGACAAAACTCACACATGTCCACCGTGCCCAGCACCTGAAGCAGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGAGCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGTCTAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGAGGCGGAGGGAGTGGCGGGGGAGGCTCTCAGATTGTCCTGAGCCAATCCCCAGCAATTCTGAGTGCTAGCCCTGGAGAGAAGGTAACAATGACTTGTCGGGCATCCCTTAGCGTCTCATCCATGCATTGGTATCAACAAAAGCCAGGTTCATCTCCAAAACCCTGGATTTACGCTACATCTAACCTGGCATCTGGGGTGCCTGCCAGATTTAGTGGATCTGGTTCCGGCACATCATATTCCCTTACAATCAGCCGAGTGGAAGCCGAGGATGCTGCAACCTATTACTGTCAACAATGGATATTTAACCCTCCCACCTTTGGGGGTGGGACTAAACTCGAAATCAAGGGCGGCTCCGAGGGCAAGAGCAGCGGCAGCGGCAGCGAGAGCAAGAGCACCGGCGGCAGCCAAGCCTATCTTCAACAATCTGGGGCTGAGCTTGTCCGGCCAGGAGCCTCCGTCAAAATGAGCTGCAAAACCTCAGGTTATACTTTTAGTAGCTATAACATGCATTGGGTAAAACAAACCCCCCGACAAGCATTGGAGTGGATAGGGGCCATATACCCCGGCAATGGAGACACAAGTTACAACCAGAAGTTTAAAGGCAAAGCTACACTCACAGTTGACAAATCCTCAAGTACTGCTTATATGCAACTCTCCTCTCTCACTTCCGAAGACAGTGCCGTATATTTTTGCACTCGGTCCAATTACTATGGATCTAGTGGCTGGTACTTTGACGTTTGGGGCACTGGGACAACTGTTACAGTGTCCAGC

1483 trispecific Ab CD3-CD20 arm of SEQ ID NO

EIVLTQSPATLSASPGERVTLSCSASSSVSYMNWYQQKPGQAPRRWIYDSSKLASGVPARFSGSGSGRDYTLTISSLEPEDFAVYYCQQWSRNPPTFGGGTKVEIKGGSEGKSSGSGSESKSTGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTRSTMHWVKQAPGQGLEWIGYINPSSAYTNYNQKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCASPQVHYDYAGFPYWGQGTLVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSQIVLSQSPAILSASPGEKVTMTCRASLSVSSMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWIFNPPTFGGGTKLEIKGGSEGKSSGSGSESKSTGGSQAYLQQSGAELVRPGASVKMSCKTSGYTFSSYNMHWVKQTPRQALEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCTRSNYYGSSGWYFDVWGTGTTVTVSS

1484 trispecific Ab CD3-CD20 arm of SEQ ID NO

GAGATCGTGCTGACCCAGTCTCCTGCCACACTGAGTGCTTCTCCAGGCGAGAGAGTGACCCTGTCCTGCTCCGCTTCCTCCTCCGTGTCCTACATGAACTGGTATCAGCAGAAGCCCGGCCAGGCTCCTCGGAGATGGATCTACGACTCTTCCAAGCTGGCCTCTGGTGTGCCAGCCAGATTTTCTGGCTCTGGCTCCGGCAGAGACTATACCCTGACCATCTCCAGCCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTGGTCTAGGAACCCTCCTACCTTTGGCGGAGGCACCAAGGTGGAAATCAAGGGCGGATCTGAGGGAAAGTCCAGCGGCTCCGGCAGCGAAAGCAAGTCCACCGGCGGAAGCCAGGTTCAACTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCTCCTCCGTCAAGGTGTCCTGCAAGGCTTCCGGCTACACCTTTACCAGATCCACCATGCACTGGGTCAAGCAGGCCCCTGGACAAGGCTTGGAGTGGATCGGCTACATCAACCCCAGCTCCGCCTACACCAACTACAACCAGAAATTCCAGGGCAGAGTGACCCTGACCGCCGACAAGTCTACCTCCACCGCCTACATGGAACTGTCCAGCCTGAGATCTGAGGACACCGCCGTGTACTACTGCGCCTCTCCTCAGGTCCACTACGACTACGCCGGCTTTCCTTATTGGGGCCAGGGCACACTGGTCACCGTTTCTTCTGAGCCCAAATCTAGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGAGCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTGTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTCCTGCGCCGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGGTTCACGCAGAAGTCTCTCTCCCTGTCTCCGGGAAAAGGAGGCGGAGGATCTGGCGGAGGTGGAAGTGGCGGAGGCGGTTCTGGTGGTGGTGGATCTCAGATCGTGCTGTCTCAGTCTCCAGCTATCCTGTCTGCTAGCCCTGGCGAGAAAGTGACCATGACCTGTAGAGCCAGCCTGTCCGTGTCCTCCATGCACTGGTATCAGCAGAAGCCTGGCAGCTCCCCTAAGCCTTGGATCTACGCCACCTCCAATCTGGCCTCTGGCGTGCCAGCTAGATTCTCCGGATCTGGCTCCGGCACCTCCTACAGCCTGACAATCTCCAGAGTGGAAGCCGAGGATGCCGCCACCTACTACTGTCAGCAGTGGATCTTCAACCCTCCTACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGGGAGGGAGCGAGGGAAAGTCCAGCGGAAGCGGCTCTGAGTCCAAATCCACCGGAGGGAGCCAGGCTTACTTGCAGCAGTCTGGTGCCGAACTCGTTAGACCTGGAGCCTCCGTGAAGATGTCCTGCAAGACCTCCGGCTACACCTTCTCCAGCTACAACATGCACTGGGTCAAGCAGACCCCTCGGCAGGCTCTGGAATGGATCGGCGCTATCTATCCTGGCAACGGCGACACCTCCTACAACCAGAAGTTCAAGGGCAAAGCTACCCTGACCGTGGACAAGTCCTCCTCCACCGCTTACATGCAGCTGTCCAGCCTGACCTCTGAGGACTCCGCCGTGTACTTCTGCACCCGGTCTAACTACTACGGCTCCTCCGGCTGGTACTTCGATGTGTGGGGAACCGGAACCACCGTGACAGTCTCTTCT

1485 trispecific Ab CD3-CD20 arm of SEQ ID NO

DIQMTQSPSSLSASVGDRVTITCRARQSIGTAIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSGSWPYTFGQGTKLEIKGGSEGKSSGSGSESKSTGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSRYNMNWVRQAPGKGLEWVSSISTSSNYIYYADSVKGRFTFSRDNAKNSLDLQMSGLRAEDTAIYYCTRGWGPFDYWGQGTLVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASLSVSSMHWYQQKPGQAPRLLIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWIFNPPTFGGGTKVEIKGGSEGKSSGSGSESKSTGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSSYNMHWVRQAPGQGLEWMGAIYPGAGDTSYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSNYYGSSGWYFDVWGKGTTVTVSS

1486 trispecific Ab CD3-CD20 arm of SEQ ID NO

GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCCGCCTCTGTGGGCGACAGAGTGACCATTACCTGCCGGGCCAGACAGTCTATCGGCACCGCTATCCACTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATTAAGTACGCCTCCGAGTCCATCTCCGGCGTGCCCTCCAGATTTTCTGGCTCTGGATCTGGCACCGACTTTACCCTGACAATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGTCAGCAGTCCGGCTCTTGGCCTTACACCTTTGGTCAGGGCACCAAGCTGGAAATCAAGGGCGGATCTGAGGGAAAGTCCAGCGGCTCCGGCAGCGAAAGCAAGTCCACCGGCGGAAGCGAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTAAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCTGCTTCTGGCTTCACCTTCAGCCGGTACAACATGAACTGGGTCCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCTCCATCTCCACCTCCAGCAACTACATCTACTACGCCGACTCCGTGAAGGGCAGATTCACCTTCTCCAGAGACAACGCCAAGAACTCCCTGGACCTGCAGATGTCTGGCCTGAGAGCTGAGGACACCGCTATCTACTACTGCACCAGAGGCTGGGGACCCTTCGATTATTGGGGCCAGGGAACCCTGGTCACCGTGTCATCTGAGCCCAAATCTAGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGAGCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTGTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGTCTCTCTCCCTGTCTCCGGGAAAAGGAGGCGGAGGATCTGGCGGAGGTGGAAGTGGCGGAGGCGGTTCTGGTGGTGGTGGATCTGAGATCGTGCTGACCCAGTCTCCAGCCACACTGTCACTGTCTCCAGGCGAGAGAGCTACCCTGTCCTGTAGAGCCTCTCTGTCCGTGTCCTCCATGCACTGGTATCAGCAGAAGCCTGGACAGGCCCCTCGGCTGCTGATCTACGCTACCTCTAATCTGGCCAGCGGTATCCCCGCCAGATTTTCTGGTTCTGGCTCTGGCACCGACTTTACCCTGACCATCTCCAGCCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTGGATCTTCAACCCTCCTACCTTTGGCGGAGGCACCAAGGTGGAAATCAAGGGAGGGAGCGAGGGAAAGTCCAGCGGAAGCGGCTCTGAGTCCAAATCCACCGGAGGGAGCCAGGTTCAACTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCTCCTCCGTGAAGGTGTCCTGCAAGGCTTCCGGCTACACCTTCTCCAGCTACAACATGCACTGGGTCCGACAGGCCCCTGGACAAGGATTGGAATGGATGGGCGCTATCTACCCTGGCGCTGGCGATACCTCTTACGCCCAGAAATTCCAGGGCAGAGTGACCATCACCGCCGACGAGTCTACCTCCACCGCCTACATGGAACTGTCCAGCCTGAGATCTGAGGACACCGCCGTGTACTACTGCGCCCGGTCTAATTACTACGGCTCCAGCGGCTGGTACTTCGACGTGTGGGGAAAGGGCACCACCGTGACAGTCTCTTCT1487 trispecific Ab CD3-CD20 arm of SEQ ID NO

EIVLTQSPATLSASPGERVTLSCSASSSVSYMNWYQQKPGQAPRRWIYDSSKLASGVPARFSGSGSGRDYTLTISSLEPEDFAVYYCQQWSRNPPTFGGGTKVEIKGGSEGKSSGSGSESKSTGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTRSTMHWVKQAPGQGLEWIGYINPSSAYTNYNQKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCASPQVHYDYAGFPYWGQGTLVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASLSVSSMHWYQQKPGQAPRLLIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWIFNPPTFGGGTKVEIKGGSEGKSSGSGSESKSTGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSSYNMHWVRQAPGQGLEWMGAIYPGAGDTSYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSNYYGSSGWYFDVWGKGTTVTVSS

1488 trispecific Ab CD3-CD20 arm of SEQ ID NO

GAGATCGTGCTGACCCAGTCTCCTGCCACACTGAGTGCTTCTCCAGGCGAGAGAGTGACCCTGTCCTGCTCCGCTTCCTCCTCCGTGTCCTACATGAACTGGTATCAGCAGAAGCCCGGCCAGGCTCCTCGGAGATGGATCTACGACTCTTCCAAGCTGGCCTCTGGTGTGCCAGCCAGATTTTCTGGCTCTGGCTCCGGCAGAGACTATACCCTGACCATCTCCAGCCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTGGTCTAGGAACCCTCCTACCTTTGGCGGAGGCACCAAGGTGGAAATCAAGGGCGGATCTGAGGGAAAGTCCAGCGGCTCCGGCAGCGAAAGCAAGTCCACCGGCGGAAGCCAGGTTCAACTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCTCCTCCGTCAAGGTGTCCTGCAAGGCTTCCGGCTACACCTTTACCAGATCCACCATGCACTGGGTCAAGCAGGCCCCTGGACAAGGCTTGGAGTGGATCGGCTACATCAACCCCAGCTCCGCCTACACCAACTACAACCAGAAATTCCAGGGCAGAGTGACCCTGACCGCCGACAAGTCTACCTCCACCGCCTACATGGAACTGTCCAGCCTGAGATCTGAGGACACCGCCGTGTACTACTGCGCCTCTCCTCAGGTCCACTACGACTACGCCGGCTTTCCTTATTGGGGCCAGGGCACACTGGTCACCGTTTCTTCTGAGCCCAAATCTAGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGAGCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTGTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGTCTCTCTCCCTGTCTCCGGGAAAAGGAGGCGGAGGATCTGGCGGAGGTGGAAGTGGCGGAGGCGGTTCTGGTGGTGGTGGATCTGAGATCGTGCTGACCCAGTCTCCAGCCACACTGTCACTGTCTCCAGGCGAGAGAGCTACCCTGTCCTGTAGAGCCTCTCTGTCCGTGTCCTCCATGCACTGGTATCAGCAGAAGCCTGGACAGGCCCCTCGGCTGCTGATCTACGCTACCTCTAATCTGGCCAGCGGTATCCCCGCCAGATTTTCTGGTTCTGGCTCTGGCACCGACTTTACCCTGACCATCTCCAGCCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTGGATCTTCAACCCTCCTACCTTTGGCGGAGGCACCAAGGTGGAAATCAAGGGAGGGAGCGAGGGAAAGTCCAGCGGAAGCGGCTCTGAGTCCAAATCCACCGGAGGGAGCCAGGTTCAACTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCTCCTCCGTGAAGGTGTCCTGCAAGGCTTCCGGCTACACCTTCTCCAGCTACAACATGCACTGGGTCCGACAGGCCCCTGGACAAGGATTGGAATGGATGGGCGCTATCTACCCTGGCGCTGGCGATACCTCTTACGCCCAGAAATTCCAGGGCAGAGTGACCATCACCGCCGACGAGTCTACCTCCACCGCCTACATGGAACTGTCCAGCCTGAGATCTGAGGACACCGCCGTGTACTACTGCGCCCGGTCTAATTACTACGGCTCCAGCGGCTGGTACTTCGACGTGTGGGGAAAGGGCACCACCGTGACAGTCTCTTCT

1489 trispecific/bispecific Ab CD79b arm HC of SEQ ID NO. 1489

QVQLQESGPGLVKPSETLSLTCSVSGASISSFYWSWIRQPADEGLEWIGRISPSGKTNYIPSLKSRIIMSLDASKNQFSLRLNSVTAADTAMYYCARGEYSGTYSYSFDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

1490 trispecific/bispecific Ab CD79b arm HC

CAGGTTCAGCTGCAAGAGTCTGGTCCTGGCCTGGTCAAGCCTTCCGAGACACTGTCTCTGACCTGCTCTGTGTCCGGCGCCTCCATCTCTTCCTTCTACTGGTCCTGGATCCGGCAGCCTGCTGACGAAGGACTGGAATGGATCGGCCGGATCAGCCCTTCTGGCAAGACCAACTACATCCCCAGCCTGAAGTCCCGGATCATCATGTCCCTGGACGCCTCCAAGAACCAGTTCTCCCTGCGGCTGAACTCTGTGACCGCTGCCGATACCGCCATGTACTACTGTGCCAGAGGCGAGTACTCCGGCACCTACTCCTACAGCTTTGACGTGTGGGGACAAGGCACCATGGTCACAGTTTCTTCTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGAGCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTGTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTCCTGCGCCGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGGTTCACGCAGAAGTCTCTCTCCCTGTCTCCGGGAAAA

1491 trispecific/bispecific Ab CD79b arm LC

DIVMTQSPLSLSVTPGEPASISCRSSESLLDSEDGNTYLDWFLQKPGQSPQLLIYTLSYRASGVPDRFSGSGSDTDFTLHISSLEAEDVGLYYCMQRMEFPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

1492 trispecific/bispecific Ab CD79b arm LC

GACATCGTGATGACCCAGTCTCCACTGAGCCTGTCTGTGACACCTGGCGAGCCTGCCTCCATCTCCTGTAGATCTTCTGAGTCCCTGCTGGACAGCGAGGACGGCAATACCTACCTGGACTGGTTCCTGCAGAAGCCCGGACAGTCTCCTCAGCTGCTGATCTACACCCTGTCCTACAGAGCCTCTGGCGTGCCCGATAGATTCTCCGGCTCTGGCTCTGACACCGACTTTACCCTGCACATCTCCAGCCTGGAAGCCGAGGATGTGGGCCTGTACTACTGTATGCAGCGGATGGAATTTCCCCTGACCTTCGGCCAGGGCACCAAGGTGGAAATCAAGCGCACCGTGGCCGCCCCTAGCGTGTTTATCTTCCCTCCCTCGGATGAGCAGCTTAAGTCAGGCACCGCATCCGTGGTCTGCCTGCTCAACAACTTCTACCCGAGGGAAGCCAAAGTGCAGTGGAAAGTGGACAACGCGCTCCAGTCGGGAAACTCCCAGGAGTCCGTGACCGAACAGGACTCCAAGGACAGCACTTATTCCCTGTCCTCCACTCTGACGCTGTCAAAGGCCGACTACGAGAAGCACAAGGTCTACGCCTGCGAAGTGACCCATCAGGGGCTTTCCTCGCCCGTGACTAAGAGCTTCAATCGGGGCGAATGC

1493 trispecific/bispecific Ab CD79b arm HC of SEQ ID NO. 1493

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYTVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCTRVDIAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

1494 trispecific/bispecific Ab CD79b arm HC

CAGGTTCAGCTGCAGCAGTCTGGCCCTGGACTGGTCAAGCCCTCTCAGACCCTGTCTCTGACCTGTGCCATCTCCGGCGACTCCGTGTCCTCTAATTCTGCCACCTGGAACTGGATCCGGCAGTCCCCTAGTAGAGGCCTGGAATGGCTGGGCAGAACCTACTACCGGTCCAAGTGGTACAACGACTACACCGTGTCCGTGAAGTCCCGGATCACCATCAATCCCGACACCTCCAAGAACCAGTTCTCCCTGCAGCTCAACAGCGTGACCCCTGAGGATACCGCCGTGTACTACTGCACCAGAGTGGATATCGCCTTCGACTACTGGGGCCAGGGCACACTGGTTACCGTTTCTTCTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGAGCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTGTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTCCTGCGCCGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGGTTCACGCAGAAGTCTCTCTCCCTGTCTCCGGGAAAA

1495 trispecific/bispecific Ab CD79b arm LC

QTVVTQPPSVSEAPRQRVTISCSGSSSNIGNHGVNWYQQLPGKAPKLLIYNDDLLPSGVSDRFSGSTSGTSGSLAISGLQSEDEADYYCAAWDDSLNGVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

1496 trispecific/bispecific Ab CD79b arm LC

CAGACAGTGGTCACCCAGCCTCCATCTGTGTCTGAGGCCCCTAGACAGAGAGTGACCATCTCCTGCTCCGGCTCCTCCTCCAACATCGGCAATCATGGCGTGAACTGGTATCAGCAGCTGCCCGGCAAGGCTCCCAAACTGCTGATCTACAACGACGACCTGCTGCCTTCTGGCGTGTCCGACAGATTCTCCGGCTCTACCTCTGGCACCTCTGGATCCCTGGCTATCTCTGGCCTGCAGTCTGAGGACGAGGCCGACTACTATTGTGCCGCCTGGGACGATTCTCTGAACGGCGTTGTGTTTGGCGGAGGCACCAAGCTGACAGTGTTGGGACAGCCTAAGGCAGCCCCCTCCGTGACCCTGTTCCCGCCATCATCCGAAGAACTGCAGGCCAACAAGGCCACGCTCGTGTGCCTGATTTCCGACTTCTACCCGGGGGCCGTGACTGTGGCCTGGAAGGCAGACTCAAGCCCTGTGAAGGCTGGCGTCGAGACTACCACCCCGTCGAAGCAATCCAACAACAAATACGCGGCGTCCAGCTACCTGAGCCTGACCCCTGAGCAGTGGAAATCCCACCGGTCCTACTCGTGCCAAGTCACCCACGAGGGATCCACTGTGGAAAAGACCGTGGCGCCGACTGAGTGTTCC

1497 trispecific/bispecific Ab CD79b arm HC

QVQLQESGPGLVKPSQTLSLTCTVSGVSISNYYWSWIRQPPGKGLEWIGRISPSGRTNYNPSLKSRVTMSLDASKNQFSLKLSSVTAADTAVYYCARGEYSGTYSYSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

1498 trispecific/bispecific Ab CD79b arm HC

CAGGTTCAGCTGCAAGAGTCTGGCCCTGGCCTGGTCAAGCCCTCTCAGACCCTGTCTCTGACCTGTACCGTGTCCGGCGTGTCCATCTCCAACTACTACTGGTCCTGGATCCGGCAGCCTCCTGGCAAAGGACTGGAATGGATCGGCCGCATCTCTCCTTCTGGTCGCACCAACTACAACCCCAGCCTGAAAAGCAGAGTGACCATGTCTCTGGACGCCTCCAAGAACCAGTTCTCCCTGAAGCTGTCCTCCGTGACCGCTGCTGATACCGCCGTGTACTACTGTGCCAGAGGCGAGTACTCCGGCACCTACTCCTACAGCTTCGACATCTGGGGCCAGGGCACCATGGTCACAGTCTCTTCTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGAGCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTGTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTCCTGCGCCGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGGTTCACGCAGAAGTCTCTCTCCCTGTCTCCGGGAAAA

1499 trispecific/bispecific Ab CD79b arm LC

DIQMTQSPSSLSASVGDRVTITCRSSQSLFDSDDGNTYLDWFQQKPGQSPKLLIQTLSYRASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQRMEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

1500 trispecific/bispecific Ab CD79b arm LC

GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCCGCCTCTGTGGGCGACAGAGTGACCATCACCTGTCGGTCCTCTCAGTCCCTGTTCGACTCTGACGACGGCAACACCTACCTGGACTGGTTCCAGCAGAAGCCCGGCCAGTCTCCTAAGCTGCTGATCCAGACACTGTCCTACAGAGCCTCTGGCGTGCCCTCCAGATTTTCCGGCTCTGGCTCTGGCACCGACTTTACCCTGACAATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGTATGCAGCGGATGGAATTTCCCCTGACCTTCGGCGGAGGCACCAAGGTGGAAATCAAGCGCACCGTGGCCGCCCCTAGCGTGTTTATCTTCCCTCCCTCGGATGAGCAGCTTAAGTCAGGCACCGCATCCGTGGTCTGCCTGCTCAACAACTTCTACCCGAGGGAAGCCAAAGTGCAGTGGAAAGTGGACAACGCGCTCCAGTCGGGAAACTCCCAGGAGTCCGTGACCGAACAGGACTCCAAGGACAGCACTTATTCCCTGTCCTCCACTCTGACGCTGTCAAAGGCCGACTACGAGAAGCACAAGGTCTACGCCTGCGAAGTGACCCATCAGGGGCTTTCCTCGCCCGTGACTAAGAGCTTCAATCGGGGCGAATGC

1501 trispecific/bispecific Ab CD79b arm LC

GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATTACCTGCAGAAGCAGCCAGAGCCTGTTCGACAGCGACGACGGCAATACCTACCTGGACTGGTTCCAGCAGAAGCCTGGCCAGAGCCCTAAGCTGCTGATCCAGACCCTGAGCTACAGAGCCAGCGGCGTGCCTAGCAGATTCTCCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCATGCAGAGAATGGAGTTCCCTCTGACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

1502 trispecific/bispecific Ab CD79b arm HC of SEQ ID NO

QVQLQESGPGLVKPSQTLSLTCTVSGVSISNYYWSWIRQPPGKGLEWIGRISPSGRTNYNPSLKSRVTMSLDASKNQFSLKLSSVTAADTAVYYCARGEYSGTYSYSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:1503 trispecific/bispecific Ab CD79b arm HC

CAGGTTCAGCTGCAAGAGTCTGGCCCTGGCCTGGTCAAGCCCTCTCAGACCCTGTCTCTGACCTGTACCGTGTCCGGCGTGTCCATCTCCAACTACTACTGGTCCTGGATCCGGCAGCCTCCTGGCAAAGGACTGGAATGGATCGGCCGCATCTCTCCTTCTGGTCGCACCAACTACAACCCCAGCCTGAAAAGCAGAGTGACCATGTCTCTGGACGCCTCCAAGAACCAGTTCTCCCTGAAGCTGTCCTCCGTGACCGCTGCTGATACCGCCGTGTACTACTGTGCCAGAGGCGAGTACTCCGGCACCTACTCCTACAGCTTCGACATCTGGGGCCAGGGCACCATGGTCACAGTCTCTTCTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGAGCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTGTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGTCTCTCTCCCTGTCTCCGGGAAAA

1499 trispecific/bispecific Ab CD79b arm LC

Table 32 provides a summary of examples of some CD79b x CD3 bispecific antibodies described herein.

TABLE 32 exemplary CD79b.times.CD3 bispecific antibodies

1504 bispecific Ab CD3 arm of SEQ ID NO

EIVLTQSPATLSASPGERVTLSCSASSSVSYMNWYQQKPGQAPRRWIYDSSKLASGVPARFSGSGSGRDYTLTISSLEPEDFAVYYCQQWSRNPPTFGGGTKVEIKGGSEGKSSGSGSESKSTGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTRSTMHWVKQAPGQGLEWIGYINPSSAYTNYNQKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCASPQVHYDYAGFPYWGQGTLVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1505 bispecific Ab CD3 arm of SEQ ID NO

GAGATCGTGCTGACCCAGTCTCCTGCCACACTGAGTGCTTCTCCAGGCGAGAGAGTGACCCTGTCCTGCTCCGCTTCCTCCTCCGTGTCCTACATGAACTGGTATCAGCAGAAGCCCGGCCAGGCTCCTCGGAGATGGATCTACGACTCTTCCAAGCTGGCCTCTGGTGTGCCAGCCAGATTTTCTGGCTCTGGCTCCGGCAGAGACTATACCCTGACCATCTCCAGCCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTGGTCTAGGAACCCTCCTACCTTTGGCGGAGGCACCAAGGTGGAAATCAAGGGCGGATCTGAGGGAAAGTCCAGCGGCTCCGGCAGCGAAAGCAAGTCCACCGGCGGAAGCCAGGTTCAACTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCTCCTCCGTCAAGGTGTCCTGCAAGGCTTCCGGCTACACCTTTACCAGATCCACCATGCACTGGGTCAAGCAGGCCCCTGGACAAGGCTTGGAGTGGATCGGCTACATCAACCCCAGCTCCGCCTACACCAACTACAACCAGAAATTCCAGGGCAGAGTGACCCTGACCGCCGACAAGTCTACCTCCACCGCCTACATGGAACTGTCCAGCCTGAGATCTGAGGACACCGCCGTGTACTACTGCGCCTCTCCTCAGGTCCACTACGACTACGCCGGCTTTCCTTATTGGGGCCAGGGCACACTGGTCACCGTTTCTTCTGAGCCCAAATCTAGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGAGCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTGTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGTCTCTCTCCCTGTCTCCGGGAAAA

SEQ ID NO:1506 bispecific Ab CD3 arm

EIVLTQSPATLSASPGERVTLSCSASSSVSYMNWYQQKPGQAPRRWIYDSSKLASGVPARFSGSGSGRDYTLTISSLEPEDFAVYYCQQWSRNPPTFGGGTKVEIKGGSEGKSSGSGSESKSTGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTRSTMHWVRQAPGQGLEWMGYINPSSAYTNYAQKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCASPQVHYDYGGFPYWGQGTLVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1507 bispecific Ab CD3 arm of SEQ ID NO

GAGATCGTGCTGACCCAGTCTCCTGCCACACTGAGTGCTTCTCCAGGCGAGAGAGTGACCCTGTCCTGCTCCGCTTCCTCCTCCGTGTCCTACATGAACTGGTATCAGCAGAAGCCCGGCCAGGCTCCTCGGAGATGGATCTACGACTCTTCCAAGCTGGCCTCTGGTGTGCCAGCCAGATTTTCTGGCTCTGGCTCCGGCAGAGACTATACCCTGACCATCTCCAGCCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTGGTCTAGGAACCCTCCTACCTTTGGCGGAGGCACCAAGGTGGAAATCAAGGGCGGATCTGAGGGAAAGTCCAGCGGCTCCGGCAGCGAAAGCAAGTCCACCGGCGGAAGCCAGGTTCAACTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCTCCTCCGTGAAAGTGTCCTGCAAGGCTTCCGGCTACACTTTTACCAGATCCACCATGCACTGGGTCCGACAGGCTCCAGGACAAGGCTTGGAGTGGATGGGCTACATCAACCCCAGCTCCGCCTACACCAACTACGCCCAGAAATTCCAGGGCAGAGTGACCCTGACCGCCGACAAGTCTACCTCCACCGCCTACATGGAACTGTCCAGCCTGAGATCTGAGGACACCGCCGTGTACTACTGCGCTTCTCCTCAGGTGCACTACGACTACGGCGGCTTTCCTTATTGGGGCCAGGGCACACTGGTCACCGTTTCTTCTGAGCCCAAATCTAGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGAGCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTGTCGAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGTCTCTCTCCCTGTCTCCGGGAAAA

In ExpiCHO-S ^TM In cells (ThermoFisher Scientific; waltham, mass., catalog number A29127), antibodies were expressed by transient transfection with purified plasmid DNA according to the manufacturer's instructions. Briefly, at 37℃8% CO ₂ And 125RPM in a rotary shaking incubator, to allow for ExpiCHO-S ^TM Cells in ExpiCHO ^TM Expression medium (ThermoFisher Scientific, catalog number A29100) was kept in suspension. After transfection to 6.0X10 ⁶ Prior to each cell/ml, the cells were passaged and diluted to maintain cell viability at 99.0% or higher. Using an ExpiFectamine ^TM CHO transfection kit (ThermoFisher Scientific, catalog No. a 29131) was transiently transfected. For each ml of diluted cells to be transfected, 0.5 microgram of bispecific antibody encoding DNA (HC 1: HC2: lc=1:2:2) and 0.5 microgram of pAdVAntage DNA (Promega, cat. No. E1711) were used, diluted to OptiPRO ^TM SFM complexing Medium. ExpiFectamine was used at a 1:4 ratio (v/v, DNA: reagent) ^TM CHO reagent, and diluted to OptiPRO ^TM Is a kind of medium. The diluted DNA was combined with transfection reagent for one minute, allowing DNA/lipid complexes to form, and then added to the cells. After overnight incubation, the ExpiCHO was incubated according to the manufacturer's standard protocol ^TM Feed and ExpiFectamine ^TM CHO enhancers are added to cells. Cells were incubated at 37℃for 7 days with rotary shaking (125 rpm) prior to harvesting the culture broth. ExpiCHO-S from transient transfection was clarified by centrifugation (30 min,3000 rcf) followed by filtration (0.2 μm PES membrane, corning; corning, N.Y.) ^TM Cell culture supernatant.

The filtered cell culture supernatant was loaded onto a pre-equilibrated (1 x dpbs, ph 7.2) MabSelect Sure protein a column (GE Healthcare) using an AKTAXpress chromatography system. After loading, the column was washed with 10 column volumes of 1x DPBS (pH 7.2). The protein was eluted with 10 column volumes of 0.1M sodium acetate (Na) (pH 3.5). 2.5M Tris HCl (pH 7.2) was added to 20% (v/v) of the volume of the eluted fraction to immediately neutralize the protein fraction. The peak fractions were pooled and loaded onto a CH1 column (thermosusher). After loading, the column was washed with 10 column volumes of 1x DPBS (pH 7.2). The protein was eluted with 10 column volumes of 0.1M sodium acetate (Na) (pH 3.5). 2.5M Tris HCl (pH 7.2) was added to 15% (v/v) of the final volume to partially neutralize the protein fraction. The high molecular weight material was removed by preparative Size Exclusion Chromatography (SEC) using Superdex 200 (GE Healthcare). After injection, the column was developed with 1 xDSL, the main peak fractions were pooled, dialyzed into 10mM histidine (pH 6.5) and filtered (0.2 μm).

The concentration of purified protein was determined by absorbance at 280nm on a Dropsense spectrophotometer. The quality of the purified protein was assessed by cSDS and analytical size exclusion HPLC (Agilent HPLC system).LAL determination using nephelometryAssociates of Cape Cod; falmouth, mass.) measures endotoxin levels.

Example 4: binding characterization of bispecific and trispecific antibodies

Bispecific CD79xCD3 antibodies at CD79 ⁺ Binding to target cells

The binding affinity of the CD79b binding arm of the CD79xCD3 bispecific molecule was assessed using cell lines that demonstrated by flow cytometry that CD79b had different endogenous expression levels on the cell surface, as shown in table 33.

CD79b antigen Density of B lymphoma cell lines

Diffuse large B cell lymphoma cell lines were incubated with CD79bxCD3 test molecules 79C3B646, 79C3B651, and 79C3B601 (1 μm initial concentration, serial dilutions of 1:3) for 1 hour at 37 ℃. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were then stained with BD staining buffer containing 1:200 dilutions of AlexaFluor 647 labeled anti-human IgG secondary antibody (Jackson Immuno; catalog No. 109-606-098) along with 1:400 dilutions of Aquas fixable Live/read stain (Invitrogen; catalog No. L34957) for 20 minutes at 4 ℃. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were analyzed using a Intellicyt (Sartorius) flow cytometer and average fluorescence intensity (MFI) was generated using Forcyt software (Sartorius). MFI was plotted using GraphPad PRISM v.8 and EC50 values were generated. The dose response curve is generated by transforming the x-axis values using the formula x=lox. The "log (agonist) versus response-variable slope (four parameters)" data were then plotted using a nonlinear regression curve fit analysis.

All CD79bxCD3 molecules showed good binding to cell lines expressing endogenous CD79B on the cell surface, with the CD79B binding arm of construct 79C3B651 showing the highest binding affinity among all tested cell lines, as shown in fig. 7A-7D and table 34.

TABLE 34 CD79bxCD3 bispecific cell binding EC50 values

⁺ ⁺ Binding of trispecific CD79xCD20xCD3 antibodies on CD79b and CD20 target cells

The binding affinity of the CD79b binding arm of CD79xCD20xCD3 trispecific molecule and control CD79bxCD3 and NullxCD20xCD3 was evaluated using cell lines that demonstrated different endogenous expression levels of CD79b and CD20 on the cell surface by flow cytometry, as shown in table 35.

CD79b and CD20 antigen Density of B lymphoma cell lines

The diffuse large B cell lymphoma cell line was incubated with CD79bxCD20xCD3 test molecules C923B74, C923B99 and C923B38, CD79xCD3 test molecules 79C3B646, 79C3B651 and 79C3B601, and NullxCD20xCD3 control molecule C923B98 (1 μm initial concentration, serial dilution at 1:3) for 1 hour at 37 ℃. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were then stained with BD staining buffer containing 1:200 dilutions of AlexaFluor647 labeled anti-human IgG secondary antibody (Jackson Immuno; catalog No. 109-606-098) along with 1:400 dilutions of Aquas fixable Live/read stain (Invitrogen; catalog No. L34957) for 20 minutes at 4 ℃. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were analyzed using a Intellicyt (Sartorius) flow cytometer and average fluorescence intensity (MFI) was generated using Forcyt software (Sartorius). MFI was plotted using GraphPad PRISM v.8 and EC50 values were generated. The dose response curve is generated by transforming the x-axis values using the formula x=lox. The "log (agonist) versus response-variable slope (four parameters)" data were then plotted using a nonlinear regression curve fit analysis.

All CD79bxCD20xCD3 molecules showed good binding to cell lines expressing endogenous CD79b and CD20 on the cell surface when compared to the binding of CD79bxCD3 and CD20xCD3 control molecules, with some trispecific constructs showing better binding affinity across the cell lines, as shown in fig. 8A-8D and tables a-10. The CD79B binding arm of trispecific construct C923B99 showed the highest binding affinity among all the tested cell lines as shown in fig. 8A to 8D and table 36.

TABLE 36 CD79bxCD20xCD3 trispecific cell binding EC50 values

⁺ Cell binding kinetics of bispecific CD79xCD3 antibodies on CD79 target cells

The binding kinetics of the CD79b binding arm of the CD79xCD3 bispecific molecule was evaluated over time using cell lines that demonstrated by flow cytometry that CD79b had different endogenous expression levels on the cell surface, as shown in table 37.

CD79b antigen Density of B lymphoma cell lines

Diffuse large B cell lymphoma cell lines were incubated with CD79bxCD3 test molecules 79C3B646, 79C3B651 and 79C3B601 (300 nM, 60nM, 12 nM) for 1 hour, 3 hours, 24 hours and 48 hours at 37 ℃. At each time point, cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were then stained with BD staining buffer containing 1:200 dilutions of AlexaFluor 647 labeled anti-human IgG secondary antibody (Jackson Immuno; catalog No. 109-606-098) for 30 minutes at 4 ℃. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were resuspended in 50 μl FACS buffer containing Cytox Green viability dye (Invitrogen, catalog No. S34860) at 1:1000 dilution. Cells were analyzed using a Intellicyt (Sartorius) flow cytometer and average fluorescence intensity (MFI) was generated using Forcyt software (Sartorius). MFI was plotted using GraphPad PRISM v.8 and EC50 values were generated.

All CD79bxCD3 bispecific constructs showed stable CD79b binding kinetics with minimal loss of signal over time, as shown in fig. 9A-9I. 79C3B651 showed excellent binding kinetics with only minimal loss of signal over time as shown in fig. 9A-9I.

⁺ ⁺ Cell binding kinetics of trispecific CD79xCD20xCD3 antibodies on CD79b and CD20 target cells

The binding kinetics of the CD79b and CD20 binding arms of the CD79xCD20xCD3 trispecific molecule were evaluated over a period of time using cell lines that demonstrated by flow cytometry that CD79b and CD20 have different endogenous expression levels on the cell surface, as shown in table 38.

CD79b and CD20 antigen Density of B lymphoma cell lines

The diffuse large B cell lymphoma cell line was incubated with CD79bxCD20xCD3 test molecules C923B74, C923B99 and C923B38, CD79xCD3 test molecules 79C3B646, 79C3B651 and 79C3B601, and NullxCD20xCD3 control molecule C923B98 (300 nM, 60nM, 12 nM) for 1 hour, 3 hours, 24 hours and 48 hours at 37 ℃. At each time point, cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were then stained with BD staining buffer containing 1:200 dilutions of AlexaFluor 647 labeled anti-human IgG secondary antibody (Jackson Immuno; catalog No. 109-606-098) for 30 minutes at 4 ℃. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were resuspended in 50 μl FACS buffer containing Cytox Green viability dye (Invitrogen, catalog No. S34860) at 1:1000 dilution. Cells were analyzed using a Intellicyt (Sartorius) flow cytometer and average fluorescence intensity (MFI) was generated using Forcyt software (Sartorius). MFI was plotted using GraphPad PRISM v.8 and EC50 values were generated.

All CD79bxCD20xCD3 bispecific constructs showed stable CD79b binding kinetics with minimal loss of signal over time, as shown in fig. 10A-10I. The trispecific construct C923B99 and the bispecific construct 79C3B651 with identical CD79B binding arm and CD20 binding arm showed excellent binding kinetics with only minimal loss of signal over time, as shown in fig. 10A to 10I.

Binding of bispecific CD79xCD3 antibodies and trispecific CD79xCD20xCD3 antibodies on pan T cells

Binding of CD3 arms of CD79xCD3 bispecific construct and CD79bxCD20xCD3 trispecific construct uses cryopreserved negative selection primary human CD3 ⁺ Pan T cells were evaluated. Primary human CD3 from four different donors ⁺ The pan T cells were incubated with CD79bxCD20xCD3 test molecules C923B74, C923B99 and C923B38 or CD79xCD3 test molecules 79C3B646, 79C3B651 (1 μm initial concentration, serial dilutions of 1:3) for 1 hour at 37 ℃. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were then stained with BD containing 1:300 dilutions of AlexaFluor 647-labeled anti-human IgG secondary antibody (Jackson Immuno; catalog No. 109-606-098) at 4 ℃ And (5) flushing and dyeing for 20 minutes. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were resuspended in 50 μl FACS buffer containing Cytox Green viability dye (Invitrogen, catalog No. S34860) at 1:1000 dilution. Cells were analyzed using a Intellicyt (Sartorius) flow cytometer and average fluorescence intensity (MFI) was generated using Forcyt software (Sartorius). MFI was plotted using GraphPad PRISM v.8. The dose response curve is generated by transforming the x-axis values using the formula x=lox. The "log (agonist) versus response-variable slope (four parameters)" data were then plotted using a nonlinear regression curve fit analysis.

All CD79bxCD20xCD3 molecules and CD79bxCD3 molecules showed moderate binding to all donor pan T cells expressing endogenous CD3 on the cell surface, as shown in fig. 11A-11D.

Example 5: functional characterization of antagonistic activity of CD79xCD3 bispecific antibodies and CD79xCD20xCD3 trispecific antibodies

⁺ ^- Bispecific CD79xCD3 and trispecific CD79xCD20xCD3 mediated targeting CD79B target cells and CD79B Cytotoxicity of target cells

The mKATE2 DLBCL target cells were maintained in complete RPMI (thermo fisher, cat. No. 11875093) 1640 medium containing 10% heat inactivated fetal bovine serum. Prior to the assay, 3-fold serial dilutions of antibodies were prepared in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum at 4-fold of the expected final concentration. The bsAb or trispecific Ab was further diluted to 200 μl by adding a mixture of target cells and effector cell suspension in a volume of 50 μl of medium per well of the 96-well plate. Target cell lines were harvested by centrifugation at 400Xg for 5 min, washed once with phenol red free RPMI 1640 medium, counted 1X 10 ⁶ The individual cells/mL density was suspended in fresh and phenol red free complete RPMI 1640 medium. Thawing healthy donor T cells (isolated by CD 3-negative selection provided by Discovery Life Sciences) in phenol red free complete medium (RPMI 1640 medium containing 10% heat inactivated fetal bovine serum) and 1X 10 after counting ⁶ Each thinThe density of cells/mL was suspended in fresh and phenol red free complete RPMI 1640 medium. The target cells and T cells were mixed to obtain a 5:1 effector cell to target cell ratio. Cell suspensions were added to the antibody dilution wells according to the plate layout (150 μl/well).

After mixing target cells and T cells with the corresponding bsAb dilutions, 80 μl was dispensed from each well (containing 200 μl of the mixture containing 10000 target cells and 50000T cells) into 384-well plates and operated in parallel. The panels were sealed using a Breathe-Easy seal. The co-cultures were then placed in an intucyte ZOOM real-time content imaging system and images were automatically acquired in two-phase and fluorescent channels every 6 hours using a 4X objective (single full aperture image) for 3 to 6 days. Target cells were detected using the optimized process definition parameters based on mKATE2 expression using the IncuCyte Zoom software. To measure the amount of target cells per well, the total red area was quantified and then the original value was derived as Excel (Microsoft Office). To quantify cancer cell killing over time, the average of each replicate was stuck in Prism (GraphPad; version 7 for PC). The Expansion Index (EI) at each time point is calculated by dividing the value at Tx by the value at T0. Growth Inhibition (GI) was calculated by normalizing each time point to the mean value of untreated wells at that time point. Area Under Curve (AUC) values for each condition were derived from the GI values. After normalization of AUC to untreated control (target and effector), antibody concentration was plotted as dose response relative to AUC values. EC50 values were generated using GraphPad PRISM v.8. The dose response curve is generated by transforming the x-axis values using the formula x=lox. The "log (agonist) versus response-variable slope (four parameters)" data were then plotted using a nonlinear regression curve fit analysis. Cytotoxicity of the lead CD79bxCD3 bispecific antibody and the CD79bxCD20xCD3 trispecific antibody (79C 3B646, C923B74, 79C3B601, C923B38, 79C3B651, C923B99, 79C3B613, C923B 98) against HBL1 cells and OCI-Ly10 cells was evaluated. The IC50 (pM) values are listed in Table 39, table 40, table 41 and Table 42.

TABLE 39 killing by HBL-1 as determined by Incucyte (average of 2 independent experiments)

Protein ID	CD79b	CD20	CD3	IC50(pM)
					79C3B645	CD9B330	NA	CD3B2089	7189.0
79C3B646	CD9B330	NA	CD3B2030	257.4
					C923B73	CD9B330	C20B22	CD3B2089	6805.0
C923B74	CD9B330	C20B22	CD3B2030	346.3
					79C3B605	CD9B374	NA	CD3B2089	29549.0
79C3B601	CD9B374	NA	CD3B2030	203.9
					C923B36	CD9B374	C20B22	CD3B2089	31040.0
C923B38	CD9B374	C20B22	CD3B2030	301.2
					79C3B650	CD9B643	NA	CD3B2089	43314.0
79C3B651	CD9B643	NA	CD3B2030	32.5
					C923B95	CD9B643	C20B22	CD3B2089	4891.0
C923B99	CD9B643	C20B22	CD3B2030	69.2

TABLE 40 OCI-Ly10 killing by Incucyte (average of 2 independent experiments)

Protein ID	CD79b	CD20	CD3	IC50(nM)
					79C3B645	CD9B330	NA	CD3B2089	18.0
79C3B646	CD9B330	NA	CD3B2030	18.3
					C923B73	CD9B330	C20B22	CD3B2089	132.4
C923B74	CD9B330	C20B22	CD3B2030	25.6
					79C3B605	CD9B374	NA	CD3B2089	54.3
79C3B601	CD9B374	NA	CD3B2030	11.7
					C923B36	CD9B374	C20B22	CD3B2089	42.0
C923B38	CD9B374	C20B22	CD3B2030	8.0
					79C3B650	CD9B643	NA	CD3B2089	7.0
79C3B651	CD9B643	NA	CD3B2030	4.7
					C923B95	CD9B643	C20B22	CD3B2089	14.8
C923B99	CD9B643	C20B22	CD3B2030	5.6

TABLE 41 CARNAVAL killing power (Incucyte)

Protein ID	CD79b	CD20	CD3	IC50(nM)
					79C3B646	CD9B330	NA	CD3B2030	1.393
C923B74	CD9B330	C20B22	CD3B2030	0.741
					79C3B601	CD9B374	NA	CD3B2030	1.645
C923B38	CD9B374	C20B22	CD3B2030	0.465
					C923B99	CD9B643	C20B22	CD3B2030	0.285

TABLE 42 Daudi killing power (Incucyte)

Protein ID	CD79b	CD20	CD3	IC50(nM)
					79C3B646	CD9B330	NA	CD3B2030	0.597
C923B74	CD9B330	C20B22	CD3B2030	0.100
					79C3B601	CD9B374	NA	CD3B2030	0.406
C923B38	CD9B374	C20B22	CD3B2030	0.071
					C923B99	CD9B643	C20B22	CD3B2030	<Testing concentration

FACS T cells are positive for a panel of target (CD79 b+ and CD20+) cell lines and negative for targets (CD 79B-and) Killing data for CD 20-) cell lines

Functional activity of the CD79bxCD3 bispecific construct and the CD79bxCD20xCD3 trispecific construct was assessed in an in vitro T cell killing assay by flow cytometry using cell lines that demonstrated by flow cytometry that CD79b and CD20 have different endogenous expression levels on the cell surface at the time point of 72 hours, as shown in table 43.

CD79b and CD20 antigen Density of B lymphoma cell lines

Target cancer cells were maintained in complete RPMI 1640 (thermo fisher, cat# 11875093) medium containing 10% heat inactivated fetal bovine serum. Prior to the assay, 3-fold serial dilutions of antibodies were prepared in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum at 4-fold of the expected final concentration. The bispecific or trispecific abs were further diluted to 200 μl by adding a mixture of target cells and effector cell suspensions, 50 μl volume of medium in each well of a 96-well plate. The target cell lines were harvested by centrifugation at 400Xg for 5 minutes and washed once with RPMI 1640 medium. Target cancer cells were targets stained with CellTrace CFSE (ThermoFisher; catalog number: C34554) at a 1/5000 dilution. Thawing healthy donor T cells (isolated by CD 3-negative selection provided by Discovery Life Sciences) in complete medium (RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum), counting followed by 1X 10 ⁶ The individual cells/mL density was suspended in fresh and phenol red free complete RPMI 1640 medium. The target cells and T cells were mixed to obtain a 5:1 effector cell to target cell ratio. Cell suspensions were added to the antibody dilution wells according to the plate layout (150 μl/well). The cells were incubated with CD79bxCD3 or CD79bxCD20xxCD3 test molecules (100 nM initial concentrationDegree, serial dilution at 1:3) were incubated together at 37℃for 72 hours. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were stained with 1:1000 dilution of fixable Live/read stain (ThermoFisher; catalog number 65-0865-14) for 15 minutes at room temperature. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were then stained with BD staining buffer containing flow panel antibodies (table 44) at 4 ℃ for 30 minutes, with the amounts of antibodies added listed in the table. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were analyzed using FACS Lyric (BD) flow cytometer and Cytobank was used to generate percent cancer cell killing. Percent cancer cell killing was plotted using GraphPad PRISM v.8 and IC50 values were generated. The dose response curve is generated by transforming the x-axis values using the formula x=lox. The "log (inhibitor) versus response-variable slope (four parameters)" data were then plotted using a non-linear regression curve fit analysis.

TABLE 44 flow panel antibodies for T cell killing assay

Antibody name	Conjugated fluorophores	Suppliers (suppliers)	Catalog number	Lot number:	amount added per well (μl)
						CD4	V500	BD Biosciences	560768	9340575	2 μl/well
CD8	PerCPCy5.5	BD Biosciences	560662	9290508	2 μl/well
						CD69	PE	BD Biosciences	560968	9049603	10 mu L/well
CD25	BV421	BD Biosciences	562443	10302	2 μl/well

In CD79b and CD20 target positive cell lines, CD79bxCD20xCD3 trispecific-mediated cytotoxicity was stronger compared to the bispecific construct. The IC50 (pM) values are listed in Table 45. No killing was observed in the target negative cell lines (fig. 12A-12B).

TABLE 45 target positivity (CARNAVAL, OCKilling power of I-Ly 10) cell line (FACS)。

Mean value of T cell mediated killing of ". 3 independent T cell donors

Mean value of T cell mediated killing of "×"4 independent T cell donors

Bispecific CD79bxCD3 mediated cytotoxicity on autologous B cells

The functional activity of the CD79bxCD3 bispecific construct was evaluated in an in vitro autologous B cell depletion assay. The functional assay utilizes PBMCs to focus on killing primary B cells and activating T cells on donor matched primary cells. Cryopreserved PBMCs from 3 different human donors were incubated with CD79bxCD3 test molecules 79C3B646, 79C3B651, and 79C3B601 (300 nM initial concentration, serial dilutions at 1:3) at 37 ℃ for 72 hours. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were stained with BD staining buffer containing 1:400 dilutions of Fc blocker (Accurate Chemical and Scientific Corp; catalog No. NB 309) and near infrared fixable Live/read stain (Invitrogen; catalog No. L10119) for 10 min at room temperature. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were then stained with BD staining buffer containing 1:100 dilutions of flow panel antibodies (Table 46) for 30 minutes at 4 ℃. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were analyzed using a Intellicyt (Sartorius) flow cytometer and average fluorescence intensity (MFI) was generated using Forcyt software (Sartorius). MFI was plotted using GraphPad PRISM v.8 and EC50 values were generated. The dose response curve is generated by transforming the x-axis values using the formula x=lox. The "log (agonist) versus response-variable slope (four parameters)" data were then plotted using a nonlinear regression curve fit analysis.

TABLE 46 flow panel antibodies for autologous B cell depletion assay

Antibody name	Conjugated fluorophores	Suppliers (suppliers)	Catalog number
				Antihuman CD25	BV650	BD Biosciences	563719
Antihuman CD4	BV510	Biolegend	317444
				Antihuman CD8	PE-Cy7	Biolegend	301012
Antihuman CD20	PE	Biolegend	302306
				Anti-human CD11c	AF647	BD Biosciences	565911
Antihuman CD2	BV605	BD Biosciences	740391

As shown in fig. 13A-13C, the CD79bxCD3 bispecific construct showed 20% drug-mediated maximum cytotoxicity with low levels of CD4 ⁺ And CD8 ⁺ T cell activation, as demonstrated by CD25 expression on these T cell subsets. The trispecific of CD79bxCD20xCD3 has a synergistic effect on drug-mediated cytotoxicity when compared to the control molecule, as shown in table 47.

Table 47.CD79bxCD20xCD3 EC50 values and maximum cytotoxicity

* Is not determined

Example 6: biophysical characterization

Determination of binding affinity by SPR

General protocol for SPR affinity assessment: affinity estimates of bispecific and trispecific constructs for human CD79b were measured by Surface Plasmon Resonance (SPR) using recombinantly expressed CD79b short and long isoform extracellular domains (CD9W7.001 and CD9W8.001, respectively) using the Biacore 8k SPR system (Biacore) at 25 ℃ and in hbsp+ buffer. Cross-reactivity of the same antibody groups against cynomolgus monkey antigen and mouse antigen (CD9W1.001 and CD9W105.001, respectively) was also assessed. Briefly, C1 sensor chips were immobilized with anti-human Fc (target immobilization level >400 RU) using the amino coupling protocol recommended by the supplier. The test antibodies were captured by immobilized anti-Fc, followed by injection of different CD79b constructs (human CD79b short and long isoforms: 30nM to 0.37nM, 3-fold dilution; cynomolgus monkey and mouse CD79b:3000nM to 37nM, 3-fold dilution) in different concentration series. The association and dissociation phases were measured for 2 or 3 minutes, respectively, and 30 minutes. The binding of the trispecific antibodies (C923B 168 and C923B 169) to CD3 was tested by injecting CD3W220.001 at a 3-fold dilution of 100nM to 1.23nM, the association and dissociation phases being measured for 3 and 15 minutes, respectively (CD 79B-00478).

The raw binding sensorgrams were treated by double reference using Biacore weight software (Biacore), and the cross-reactivity of the treated sensorgrams was analyzed and fitted to a 1:1langmuir model to obtain on-rate, off-rate and affinity.

SPR binding results: as shown in tables 48 and 49, these bispecific and trispecific antibodies bind to the human CD79b long isoform (hu CD79b long) with an affinity of 0.02nM to 0.06nM and bind to the CD79b short isoform (hu CD79b short) with an affinity between 0.27nM to 0.64 nM. The antibody group showed very poor cross-reactivity (KD estimation >3000 nM) to cynomolgus monkey CD79b, or did not bind to mouse CD79 b. C923B168 binds the recombinant CD3 antigen with an affinity of 0.5 nM. For those constructs with complex kinetic binding profiles using the indicated antigens, no quantitative kinetics/affinities are reported, as indicated in the summary table below.

TABLE 48 binding affinity of bispecific antibody constructs

* No sample submitted for SPR binding analysis

* *: since Cris7 b-derived CD3 antibodies were observed to have a complex SPR binding spectrum (historically observed knots

Fruit), the affinity for CD3 was not determined.

TABLE 49 binding affinity of trispecific antibody constructs

* No sample submitted for SPR binding analysis

* Affinity for CD20 or CD3 was not determined due to SPR constraints on CD20 nano-phospholipid discs or the complex binding spectra observed for the Cris7 b-derived CD3 antibodies (historically observed results).

Determination of binding epitopes by HDX-MS

The CD79B epitope bound by the trispecific molecules CD9B374 and CD9B643 was mapped by hydrogen deuterium exchange mass spectrometry (HDX-MS) according to the following protocol.

General procedure for HDX-MS data acquisition. HDX-MS sample preparation was performed using an automated HDx system (LEAP Technologies, morrisville, NC). The column and pump are: protease, type XIII protease (type XIII protease from Aspergillus zoffii)/pepsin column (w/w, 1:1;2.1 mm. Times.30 mm) (NovaBioAssays Inc., woburn, mass.); wells, ACQUITY UPLC BEH C VanGuard pre-column (2.1X5 mm) (Waters, milford, mass.), analysis, accumore C18 (2.1X100 mm) (Thermo Fisher Scientific, waltham, mass.); and LC pump, VH-P10-A (Thermo Fisher Scientific). The loading pump (from the protease column to the trap column) was set with 0.1% formic acid aqueous solution at a flow rate of 600. Mu.L/min. The gradient pump (from trap column to analytical column) was set to gradient from 0.1% aqueous formic acid in 9% acetonitrile to 0.1% aqueous formic acid in 35% acetonitrile at a flow rate of 100 μl/min over 20 minutes.

MS data acquisition Using LTQ ^TM Orbitrap Fusion Lumos Mass Spectrometry (Thermo Fisher Scientific) with capillary temperature 275 ℃, resolution 120,000 and mass range (m/z) 300-1,800.

HDX-MS data extraction. Peptide identification of non-deuterated samples was performed prior to HDX experiments using BioPharma Finder 3.0 (Thermo Fisher Scientific). Centroid values were extracted from MS raw data files of HDX experiments using hdexarner version 2.5 (Sierra analysis, modesto, CA).

HDX-MS data analysis. The extracted HDX-MS data were further analyzed in Excel. All exchange time points (at pH 6.4 or pH 7.4, at 3.2 ℃) were converted to equivalent time points at pH 7.4 and 23 ℃.

Results

HDX-MS analysis showed that CD9B374 and CD9B643 bind to nearly identical conformational epitopes of CD79 consisting of the following residues: residues 30 to 42 (SEDRYRNPKGSAC; SEQ ID NO: 253), residues 50 to 52 (PRF), residues 81 to 86 (EMENP; SEQ ID NO: 254), and residues 144 to 148 (GFSTL; SEQ ID NO: 255). The residue number is the number of cd9b_human (P40259).

Thermal stability of trispecific CD79bxCD20xCD3 antibodies as determined by DSC and DSF

The thermal stability of C923B168 and C923B169 was determined by Differential Scanning Calorimetry (DSC) and Differential Scanning Fluorescence (DSF).

In this characterization, tonset and Tagg are determined by DSF, while other thermal stability transitions of Tm are determined by DSC. As shown in table 50, C923B168 and C923B169 had good thermal stability, tonset >61 ℃ and Tm1>65 ℃.

TABLE 50 transition temperature of trispecific CD79bxCD20xCD3 antibodies：

Example 7: functional characterization of CD79xCD20xCD3 trispecific antibodies

Binding of trispecific CD79bxCD20xCD3 antibodies to pan T cells

Binding of the CD3 arm of the CD79bxCD20xCD3 trispecific construct uses cryopreserved negative selection primary human CD3 ⁺ Pan T cells were evaluated. Primary human CD3 from three different donors ⁺ Thin pan TCells were incubated with CD79bxCD20xCD3 test molecules C923B169 and C923B168 (1. Mu.M initial concentration, serial dilutions at 1:3) for 1 hour at 37 ℃. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were then stained with BD staining buffer containing 1:300 dilutions of AlexaFluor 647 labeled anti-human IgG secondary antibody (Jackson Immuno; catalog No. 109-606-098) for 20 minutes at 4 ℃. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were resuspended in 50 μl FACS buffer containing Cytox Green viability dye (Invitrogen, catalog No. S34860) at 1:1000 dilution. Cells were analyzed using a Intellicyt (Sartorius) flow cytometer and average fluorescence intensity (MFI) was generated using Forcyt software (Sartorius). MFI was plotted using GraphPad PRISM v.8. The dose response curve is generated by transforming the x-axis values using the formula x=lox. The "log (agonist) versus response-variable slope (four parameters)" data were then plotted using a nonlinear regression curve fit analysis.

As shown in table 51, all CD79bxCD20xCD3 molecules were shown to bind to all donor pan T cells expressing endogenous CD3 on the cell surface.

TABLE 51 binding of C923B169 and C923B168 CD79bxCD20xCD3 to pan CD 3T cells。

Ud=undetermined

⁺ ⁺ Data on killing of FACS T cells on a set of target-positive (CD 79b and CD 20) cell lines

Functional activity of the CD79bxCD20xCD3 trispecific construct was assessed in an in vitro T cell killing assay by flow cytometry using cell lines that demonstrated by flow cytometry that CD79b and CD20 had different endogenous expression levels on the cell surface at time points of 48 hours and 72 hours, as shown in table 52.

Watch 52CD79B and CD20 antigen Density of B lymphoma cell lines

Target cancer cells were maintained in complete RPMI-1640 (thermo fisher, cat# 11875093) medium containing 10% heat inactivated fetal bovine serum. Prior to the assay, 3-fold serial dilutions of antibodies were prepared in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum at 4-fold of the expected final concentration. The bispecific or trispecific abs were further diluted to 200 μl by adding a mixture of target cells and effector cell suspensions, 50 μl volume of medium in each well of a 96-well plate. The target cell lines were harvested by centrifugation at 400Xg for 5 minutes and washed once with RPMI 1640 medium. Target cancer cells were targets stained with CellTrace CFSE (ThermoFisher; catalog number: C34554) at a 1/5000 dilution. Thawing healthy donor T cells (isolated by CD 3-negative selection provided by Discovery Life Sciences) in complete medium (RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum), counting followed by 1X 10 ⁶ The individual cells/mL density was suspended in fresh and phenol red free complete RPMI 1640 medium. The target cells and T cells were mixed to obtain a 5:1 effector cell to target cell ratio. Cell suspensions were added to the antibody dilution wells according to the plate layout (150 μl/well). Cells were incubated with CD79bxCD20xxCD3 test molecules C923B169 and C923B168 (100 nM initial concentration, serial dilutions at 1:3) for 48 and 72 hours at 37 ℃. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were stained with 1:1000 dilution of fixable Live/read stain (ThermoFisher; catalog number 65-0865-14) for 15 minutes at room temperature. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were then stained with BD staining buffer containing flow panel antibodies (table 53) at 4 ℃ for 30 minutes, with the amounts of antibodies added listed in the table. All cells were stained with BD staining buffer (BD Biosciences; catalogue554657), centrifuged at 1200RPM for 3 minutes, and the supernatant discarded. Cells were analyzed using FACS Lyric (BD) flow cytometer and Cytobank was used to generate percent cancer cell killing. Percent cancer cell killing was plotted using GraphPad PRISM v.8 and IC50 values were generated. The dose response curve is generated by transforming the x-axis values using the formula x=lox. The "log (inhibitor) versus response-variable slope (four parameters)" data were then plotted using a non-linear regression curve fit analysis.

TABLE 53 flow panel antibodies for T cell killing assay

CD79bxCD20xCD3 trispecific-mediated potent cytotoxicity. IC50 (nM) values and maximum killing values are listed in tables 54 and 55.

TABLE 54C 923B169 and C923B168CD79bxCD20xCD3 were positive for target at 48 hours (CARNAVAL, OCI-Ly10, JAK 0-1) cell lines. IC50 (nM) and percent maximum killing are listed in the table. Come to Mean from 2 independent T cell donors。

TABLE 55C 923B169 and C923B168CD79bxCD20xCD3 were positive for target at 72 hours (CARNAVAL, OCI-Ly10, JAK 0-1) cell lines. IC50 (nM) and percent maximum killing are listed in the table. Come to Mean from 2 independent T cell donors。

Cytotoxicity of autologous B cells mediated by C923B169 and C923B168CD79bxCD20xCD3

Functional activity of the C923B169 and C923B168CD79bxCD20xCD3 constructs was assessed in an in vitro autologous B cell depletion assay. The functional assay utilizes PBMCs to focus on killing primary B cells and activating T cells on donor matched primary cells. Cryopreserved PBMCs from 3 different human donors were incubated with CD79bxCD20xCD3 test molecules C923B169 and C923B168 (300 nM initial concentration, serial dilutions at 1:3) at 37 ℃ for 72 hours. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were stained with BD staining buffer containing 1:400 dilutions of Fc blocker (Accurate Chemical and Scientific Corp; catalog No. NB 309) and near infrared fixable Live/read stain (Invitrogen; catalog No. L10119) for 10 min at room temperature. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were then stained with BD staining buffer containing 1:100 dilution of flow panel antibody (table 56) for 30 minutes at 4 ℃. All cells were washed with BD staining buffer (BD Biosciences; catalog number 554657), centrifuged at 1200RPM for 3 minutes, and the supernatant was discarded. Cells were analyzed using a Intellicyt (Sartorius) flow cytometer. EC50 values were generated using GraphPad PRISM v.8. The dose response curve is generated by transforming the x-axis values using the formula x=lox. The "log (agonist) versus response-variable slope (four parameters)" data were then plotted using a nonlinear regression curve fit analysis.

TABLE 56 flow panel antibodies for autologous B cell depletion assay

The CD79bxCD20xCD 3C 923B169 and C923B168 constructs showed drug-mediated maximum cytotoxicity (table 57) of 69% to 95% with low levels of CD4 ⁺ And CD8 ⁺ T cell activation, as demonstrated by CD25 expression on these T cell subsets.

Table 57C 923B169 and C923B168 CD79bxCD20xCD3 at 72 hours in the primary autologous B cell depletion assay Killing of B cells. EC50 (nM) and percent maximum killing are listed in the table. Listed are T cells from 3 independent cells Value of donor。

Example 8: generation of bispecific PSMAxCD3 antibodies

Example 8.1: FAB arm exchange of anti-PSMA and anti-CD 3 antibodies

The formation of bispecific antibodies requires two parent monoclonal antibodies (mabs), one specific for the targeting arm (e.g., PMSA) and one specific for the effector arm (e.g., CD 3). The selected monospecific anti-PSMA and anti-CD 3 antibodies were expressed as IgG1/κ engineered to have L234A, L235A and D265S substitutions (EU numbering) for cF silencing. The monospecific anti-PSMA and anti-CD 3 antibodies selected were also expressed as IgG4 antibodies. Mutations designed to promote selective heterodimerization of the Fc domain are also engineered in the Fc domain.

Under the CMV promoter, the monospecific antibody was expressed in CHO cell lines as described above). The parent PSMA and CD3 antibodies were purified using protein a column with elution buffer, i.e., 100mM NaAc (pH 3.5), and neutralization buffer, i.e., 2M Tris (pH 7.5) and 150mM NaCl. anti-PSMA and anti-CD 3 monoclonal antibodies were dialyzed into D-PBS, pH 7.2 buffer.

For the DuoBody antibody, after purification of the parent monospecific antibody, the bispecific PSMAxCD3 antibody was generated by mixing the parent PSMA antibody with the desired parent CD3 antibody under reducing conditions in 75mM cysteamine-HCl and incubating overnight at room temperature for Fab arm exchange in vitro, as in international patent publication No. WO 2011/131746. The recombination reaction is based on a molar ratio wherein a set amount of PSMA antibody (e.g., 10mg or about 74.6 nanomolar) is combined with CD3 antibody (e.g., about 70.87 nanomolar), wherein the amount of PSMA antibody added is 5% higher than the CD3 antibody. The concentration of PSMA antibody stock varied between 0.8mg/mL to 6mg/mL, and the volume of the recombination reaction was different in each pair. The recombinants were then dialyzed overnight against PBS to remove the reducing agent. The PSMAxCD3 bispecific antibody reaction was performed with an excess of PSMA antibody (ratio) to minimize the amount of unreacted CD3 parent antibody remaining after recombination.

Other bispecific antibodies are produced by cotransfection of HC1:HC2:LC2, typically with a DNA ratio of 1:1:3. By protein a chromatography and CH1 affinity capture, followed by purification by ion exchange based chromatography.

Exemplary PSMAxCD3 multispecific antibodies are provided in tables 58-63.

Psma×cd3 bispecific antibody: clone description

Psma×cd3 bispecific antibody: CD3 arm description

Psma×cd3 bispecific antibody: PSMA arm description

Table 61 psma×cd3 bispecific antibodies: clone description

Table 62 psma×cd3 bispecific antibodies: CD3 arm description

Table 63 psma×cd3 bispecific antibodies: PSMA arm description

Example 8.2: analytical characterization of bispecific anti-PSMAxCD 3 antibodies

The protein concentration of each purified bispecific Ab was determined by measuring absorbance at 280nm on a NANODROP1000 spectrophotometer or Trinean DROPSENSE-96 multi-channel spectrophotometer and calculated using the extinction coefficients based on the amino acid sequence. SE HPLC of purified antibodies was performed by: samples were run on TOSOH TSKgel BioAssist G SWxl column in 0.2M sodium phosphate at pH 6.8 at a rate of 1 mL/min for 20 minutes on Waters Alliance HPLC. The column effluent was monitored with absorbance at 280 nm. anti-PSMA-CD 3 bispecific antibodies were further analyzed by whole mass analysis to determine the proper formation of heterodimers.

Example 9: epitope mapping of anti-PSMAxCD 3 antibodies

Example 9.1: HDX-MS epitope mapping

The epitopes of the two anti-PSMA/CD 3 bispecific antibodies PS3B1352 and PS3B1353 were determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS). See fig. 14. Human PSMA antigen was used for epitope mapping experiments. See fig. 15.

Exchange experiments with HDX-MS briefly, 10. Mu.L of 6.0. Mu.M human PSMA with 30. Mu. L H with or without 7.3. Mu.M antibody ₂ O or deuterated buffer (20 mM MES, pH 6.4, 95% D of 150mM NaCl ₂ O solution, or 20mM Tris, pH 8.4, 95% D of 150mM NaCl ₂ O solution) to begin the exchange reaction. The reaction mixtures were incubated at 23℃for 15s, 50s, 150s, 500s or 1,500s and then quenched at the different time points by the addition of chilled 40. Mu.L 8M urea, 1M TCEP (pH 3.0). The quenched solution was analyzed immediately.

General procedure for HDX-MS data acquisition. HDX-MS sample preparation was performed using an automated HDx system (LEAP Technologies, morrisville, NC). The column and pump are: protease, type XIII protease (type XIII protease from Aspergillus zoffii (Aspergillus saitoi)/pepsin column (w/w, 1:1;2.1 mm. Times.30 mm) (NovaBioAssays Inc., woburn, mass.); wells, ACQUITY UPLC BEH C VanGuard pre-column (2.1X5 mm) (Waters, milford, mass.), analysis, accumore C18 (2.1X100 mm) (Thermo Fisher Scientific, waltham, mass.); and LC pump, VH-P10-A (Thermo Fisher Scientific). The loading pump (from protease column to trap column) was set with 99% water, 1% acetonitrile and 0.1% formic acid at a flow rate of 600 μl/min. The gradient pump (from trap column to analytical column) was set to gradient from 8% acetonitrile in 0.1% formic acid in water to 28% acetonitrile in 0.1% formic acid in water at a flow rate of 100 μl/min over 20 minutes.

MS data acquisition. Using LTQ ^TM Orbitrap Fusion Lumos Mass Spectrometry (Thermo Fisher Scientific) where the capillary temperature is 275 ℃, the resolution is 150,000 and the mass range (m/z) is 300-2,000.

HDX-MS data extraction. Peptide identification of non-deuterated samples was performed prior to HDX experiments using BioPharma Finder 2.0 (Thermo Fisher Scientific). Centroid values were extracted from MS raw data files of HDX experiments using HDExaminer version 2.4 (Sierra analysis, modesto, CA).

Example 10: characterization of bispecific anti-PSMAxCD 3 antibodies

Example 10.1: binding affinity of bispecific anti-PSMAxCD 3 antibodies to human PSMA

The binding affinity of anti-PSMA to recombinant human, cynomolgus monkey or mouse PSMA was determined by Surface Plasmon Resonance (SPR) using a Biacore 8K instrument. Antibodies were captured on goat anti-Fc antibody modified C1 chips and titrated with 3-fold serial dilutions of PSMA antigen ranging in concentration from 100nM to 11.1 nM. Association and dissociation were monitored for 3 minutes and 15 minutes, respectively, using a flow rate of 50 μl/min. The raw binding data was referenced by subtracting the analyte binding signal from the blank and analyzed using Biacore Insight assessment software using a 1:1langmuir binding model to obtain kinetics for calculation of binding affinity. Kd data are summarized in table 64. anti-PSMA was captured using anti-human Fc antibodies and the antigen was injected into the solution.

Table 64: interaction of anti-PSMAxCD 3 bispecific antibodies with human PSMA obtained by BIACORE (SPR) method Affinity for use (KD)。

The binding affinity of the anti-PSMA antibodies to recombinant human PSMA was determined by Surface Plasmon Resonance (SPR) using a BIACORE 8K instrument (ELN PSMA-00702). Antibodies were captured on goat anti-Fc antibody modified C1 chips and titrated with 3-fold serial dilutions of PSMA antigen ranging in concentration from 1nM to 100nM human PSMA. Association and dissociation were monitored for 3 minutes and 30 minutes, respectively, using a flow rate of 50 μl/min. The raw binding data were referenced by subtracting the analyte binding signal from the blank and analyzed by Biacore Insight evaluation software using a 1:1langmuir binding model to obtain kinetics for calculation of binding affinity. Kinetic parameters for binding to selected antibodies are shown in table 65. anti-PSMA was captured using anti-human Fc antibodies and the antigen was injected into the solution.

Table 65: affinity of anti-PSMA antibodies obtained by Biacore (SPR) method for interaction with human PSMA (KD)。

The binding affinity of anti-PSMA to recombinant human or cynomolgus PSMA was determined by Surface Plasmon Resonance (SPR) using a Biacore 8K instrument (ELN PSMA-00721). Antibodies were captured on goat anti-Fc antibody modified C1 chips and titrated with 3-fold serial dilutions of PSMA antigen ranging in concentration from 100nM to 3.7nM (human PSMA) or from 100nM to 3.7nM, or from 22.2nM to 600nM (cynomolgus PSMA). Association and dissociation were monitored for 3 minutes and 60 minutes, respectively, using a flow rate of 50 μl/min. The raw binding data were referenced by subtracting the analyte binding signal from the blank and analyzed by Biacore Insight evaluation software using a 1:1langmuir binding model to obtain kinetics for calculation of binding affinity. Kinetic parameters for binding to selected antibodies are shown in table 66. anti-PSMA was captured using anti-human Fc antibodies and the antigen was injected into the solution.

Table 66: mutual anti-PSMA antibodies obtained by Biacore (SPR) method with human, cynomolgus monkey or mouse PSMA Affinity for action (KD) ×。

* The average value of the data in line 1 is 3. The average value of the data in the other rows is 2.

Example 10.2: thermal stability of bispecific anti-PSMAxCD 3 antibodies

The thermostability (conformational stability information, including Tm and Tagg) of the anti-PSMAxCD 3 antibodies was determined by the nanoDSF method as described above using a promethaus instrument. Briefly, measurements were made by loading samples from 384 Kong Yangben plates into 24-well capillaries. And (5) repeating the operation. The thermal scan range was 20 ℃ to 95 ℃ at a rate of 1.0 ℃/min. The data were processed to obtain 330nm, 350nm, integrated data for the ratio 330/350 and first derivative analysis, and scatter data for thermal transitions, unfolding onset, tm, and Tagg from them, summarized in table 67.

Table 67: thermal stability data for bispecific anti-PSMAxCD 3 antibodies obtained using nanoDSF instruments。

Name of the name	T _agg	T _m 1	T _m 2	T _m 3
					PS3B917	68.2℃	63.5℃	68.8℃
PS3B918	67.8℃	63.4℃	68.2℃
					PS3B913	75.1℃	63.4℃	76.3℃
PS3B915	69.2℃	63.5℃
					PS3B914	64.5℃	61.9℃	67.0℃	75.8℃
PS3B916	59.1℃	59.9℃
					PS3B919	81.0℃	63.5℃	68.3℃	85.7℃
PS3B921	78.9℃	63.5℃	80.8℃
					PS3B920	82.6℃	63.4℃	87.5℃
PS3B922	80.5℃	63.3℃	83.4℃
					PS3B912	77.1℃	63.6℃	78.4℃
PS3B930	72.2℃	70.1℃
					PS3B931	72.2℃	69.5℃
PS3B926	75.6℃	70.2℃	75.6℃
					PS3B928	72.2℃	69.1℃
PS3B927	69.1℃	66.5℃	69.4℃
					PS3B929	69.1℃	58.0℃	68.0℃
PS3B932	82.7℃	70.4℃	85.8℃
					PS3B934	79.3℃	70.4℃	80.9℃
PS3B933	83.7℃	70.0℃	87.8℃
					PS3B935	81.5℃	70.2℃	83.7℃
PS3B925	77.4℃	70.3℃	77.9℃

Example 10.3: binding of bispecific PSMAxCD3 antibodies on psma+ cells

The ability of selected bispecific PSMAxCD3 antibodies to bind to PSMA-expressing prostate cancer cell lines was evaluated.

22RV1 and C4-2B cells were seeded at 50,000 cells/well in 50. Mu.l of assay medium (RPMI, 10% HI FBS) in a V-floor. Serial dilutions of the antibodies were prepared in assay medium and 50 μl of the antibody dilution was added to the cell-containing plates. Plates were incubated at 37℃for 60min, at which time 100. Mu.l of staining buffer (Becton Dickinson catalog number 554657) was added to all wells of each plate. Plates were centrifuged at 300 XG for 5 min and the medium removed from the wells. 200 μl of staining buffer was added to all wells of each plate. Plates were centrifuged at 300 XG for 5 min and the medium removed from the wells. Mu.l of 2. Mu.g/ml AlexaFluor 647-labeled goat anti-human Fc was added to all wells of the plates and the plates were incubated at 4℃for 30 min. Mu.l of staining buffer was added to all wells of each plate. Plates were centrifuged at 300 XG for 5 min and the medium removed from the wells. 200 microliters of running buffer (staining buffer plus 1mM EDTA,0.1% pluronic acid) was added to all wells of the plate. Plates were centrifuged at 300 XG for 5 min and the medium removed from the wells. Thirty microliters of running buffer was added to all cell-containing wells and the plates were analyzed on an IQUE Plus instrument (Sartorius). Briefly, cells are gated on FCS and SSC gates to eliminate cell debris, and then cell populations are gated on singlet cells. Antibody binding was assessed in the red laser channel. The signal (Mab plus secondary antibody) to background (secondary antibody only) ratio was calculated for each plate and the resulting data plotted against bispecific antibody concentration in GeneData Screener using a 4-parameter curve fit to generate EC50 values, the results summarized in table 68.

Table 68: bispecific PSMA x CD3 antibodies that bind to PSMA-expressing cell lines in flow cytometry assays EC50 value of (2)。

Binding on pan T cells by mobilized anti-PSMA/CD 3 bispecific. Human pan T cells (Biological Specialty Corporation, colmar, PA) were thawed and transferred to 15mL Erlenmeyer flasks with DPBS. The cells were centrifuged at 1300rpm for 5 minutes. The DPBS was aspirated and the cells resuspended in DPBS. Cells were counted using a Vi-cell XR cell viability analyzer and seeded at 100K/well in 100uL DPBS. The plates were centrifuged at 1200rpm for 3 minutes and washed 2 times with DPBS. Cells were stained with Violet Live/read dye (Thermo-Fisher) and incubated at room temperature in the dark for 25min. Cells were centrifuged and washed 2 times with FACS staining buffer (BD Pharmingen). Test antibodies were diluted in FACS staining buffer to a final starting concentration of 1 μm, and 3-fold serial dilutions were prepared from the starting concentration for a total of 10 dilution points. Serial dilutions of test antibodies (100 μl/well) were added to the cells and incubated for 30min at 37 ℃. Cells were washed 2 times with FACS staining buffer and AlexaFluor 647 conjugated donkey anti-human secondary antibody (Jackson Immunoresearch) was added and incubated with cells for 30min at 4 ℃. Cells were washed 2 times with FACS staining buffer and resuspended in 100 μl FACS buffer. Cells were run on BD Celesta using FACSDiva software and analyzed using FLOWJO. FIG. 16 shows that PSMA/CD3 bispecific antibodies detected by flow cytometry showed differential CD3 cell binding spectra.

After incubation in RPMI medium plus 10% fetal bovine serum for 1 hour at 37 ℃, a binding curve was generated for the prostate cell line C4-2B, shown in table 69 below and figure 17. At 3-fold dilution, the molecular concentration of 12 spots ranged from 500nM to 0nM. Selective binding to PSMA was verified using isotype control. The values reported in Table 69 were obtained by fitting the data to a four parameter function of ligand binding to generate values for y-min, y-max, EC50 and Hill. EC90 was calculated using the equation EC90 = (90- (100-90)) ≡1/Hill EC 50. All curves exhibited similar Y-min, with an average of all curves of 7.1+/-1.3E+4. See table 69. None of the Y-min values deviate significantly from the average. The average fit Y-max value is 1.7+/-0.6E+6. The molecule PSMB1069 exhibited a binding signal 2-fold higher than the average. None of the other molecules showed significant differences from the average. These molecules showed an average EC50 = 17+/-12nM.

Table 69: anti-PSMA antibodies that bind to PSMA-expressing cell line C4-2B as measured by flow cytometry assays EC50 value of (2)。

Binding of anti-PSMA variant/CD 3 bispecific antibodies on T cells by flow cytometry. The C4-2B human prostate tumor cells were washed with DPBS and 0.25% trypsin was added to isolate the cells. Media was added to neutralize trypsin and cells were transferred to 15mL Erlenmeyer flasks with DPBS. The cells were centrifuged at 1300rpm for 5 minutes. The DPBS was aspirated and the cells resuspended in DPBS. Cells were counted using a Vi-cell XR cell viability analyzer and seeded at 100K/well in 100. Mu.L of DPBS. The plates were centrifuged at 1200rpm for 3 minutes and washed 2 times with DPBS. Cells were stained with Violet Live/read dye (Thermo-Fisher) and incubated at room temperature in the dark for 25min. Cells were centrifuged and washed 2 times with FACS staining buffer (BD Pharmingen). Test antibodies were diluted in FACS staining buffer to a final starting concentration of 100nM, and 3-fold serial dilutions were prepared from the starting concentration for a total of 10 dilution points. Serial dilutions of test antibodies (100 μl/well) were added to the cells and incubated for 30min at 37 ℃. Cells were washed 2 times with FACS staining buffer and AlexaFluor 647 conjugated donkey anti-human secondary antibody (Jackson Immunoresearch) was added and incubated with cells for 30min at 4 ℃. Cells were washed 2 times with FACS staining buffer and resuspended in 100 μl FACS buffer. Cells were run on BD CELESTA using FACSDiva software and analyzed using FLOWJO. Fig. 18 shows that PSMA/CD3 bispecific antibodies detected by flow cytometry showed similar PSMA cell binding spectra.

Example 10.4: internalization of PSMA

The C4-2B human prostate tumor cells were washed with DPBS and 0.25% trypsin was added to isolate the cells. Media was added to neutralize trypsin and cells were transferred to 15mL Erlenmeyer flasks with DPBS. The cells were centrifuged at 1300rpm for 5 minutes. The DPBS was aspirated and the cells resuspended in DPBS. Cells were counted using a Vi-cell XR cell viability analyzer and seeded at 40K/well50 μl of PRMI+10% HI FBS without phenol red. PSMA/CD3 bispecific antibody or control antibodyThe human Fab-fluor-pH red antibody labeling dye was incubated for 15 minutes, then 50. Mu.L of conjugated PSMA/CD3:Fab-fluor-pH red complex was added to wells containing C4-2B cells. The plates were placed at 37℃in an IncuCyte +.5% CO2>(Essen) for 24 hours. Treatment of Ab internalized by target cells with acid lysosomes Fab-fluor complexes generates a red fluorescent signal consisting of +.>Capturing and analyzing. Figure 19 shows PSMA/CD3 bispecific antibody internalization, but at a lower rate than transferrin receptor.

Example 10.5: measurement of T cell mediation of psma+ cells by bispecific PSMAxCD3 antibodies via flow cytometry Killing power of guide

The ability of selected bispecific PSMA x CD3 antibodies to mediate T cell mediated killing of prostate cancer cells was assessed.

T cell mediated killing of PSMA x CD3 bispecific antibodies was measured using an assay that indirectly measures cell killing by flow cytometry. Target cell populations were identified based on cell viability test samples and controls were prepared at 20nM in assay medium (10% RPMI,10% HI FCS). Semi-log serial dilutions were prepared for 11 point titration of compounds in sterile polypropylene plates. Additional wells were used for compound-free controls, wells containing T cells or tumor cells only in the assay medium. C4-2B cells were harvested from the cell culture flask and resuspended in PBS. Cells were stained with 20pM CFSE for 10 min at room temperature. 25mL of HI FBS was added to terminate the staining reaction, and the cells were centrifuged at 300 XG for 5 minutes. Diluting cells to 1X 10 ⁶ Per ml, and then as tumor target cells in 50. Mu.L assay medium50,000 cells/well were inoculated into V-bottom tissue culture treated polystyrene assay plates. 50. Mu.L/well of assay medium was added to control wells that did not receive tumor cells. Human pan T cell vials were thawed in a water bath set to 37 ℃ and washed twice by adding 10ml of assay medium and centrifuging at 400 x G for 5 minutes. T cells were resuspended in assay medium to 1×10 ⁶ Per mL, and 50 μl of assay medium containing 50,000/well was added to the assay plate containing tumor target cells. mu.L/well assay medium was added to control wells that did not receive tumor cells. Serial dilutions of 100 μl/well of antibody were added to assay plates containing cell mixtures of target cells and effector cells. The plates were incubated at 37℃with 5% CO ₂ Incubate in a humidified cell incubator for 72 hours.

After incubation, the assay plates were centrifuged at 500 XG for 5 minutes and the medium removed from the wells. 150 μl of DPBS was added to each well and the plate was centrifuged at 500×g for 5 minutes and the medium removed from the well. Viable tumor cells in culture were assessed using flow cytometry on INTELLICYT IQ Plus using near infrared live/dead staining. T cell activation was assessed using a light violet labeled anti-CD 25 MAB. Cells were gated in FSC and SSC gates to eliminate debris. Tumor cells were identified as CFSE positive cells. T cells were identified as CSFE negative cells. Tumor cell viability was calculated as the number of live/dead stain positive tumor cells as a percentage of total CSFE cells. Activated T cells were calculated as the percentage of the number of CD25 positive cells to the total number of viable CFSE negative populations. Percent data for dead tumor cells and activated T cells were plotted against antibody concentration in Gene Data Screener using a 4-parameter curve fit to generate EC50 values. Table 70 shows EC50 values for T cell activation and tumor cell killing.

Table 70: in vitro T cell mediated killing of tumor cells by bispecific PSMA x CD3 antibodies and antibodies Induced T cell activation。

Example 10.6: measurement of T cell mediation of PSMA+ cells by bispecific PSMA AxCD3 antibodies via INCUCYTE Is of killing power of (2)

By being based onIs evaluated for the ability of selected bispecific PSMAxCD3 antibodies to mediate T cell mediated killing of prostate cancer cells.

Healthy donor T cells. psma+c4-2B cells stably expressing red nuclear dye were generated for use in IncuCyte-based cytotoxicity assays. Frozen vials of healthy donor T cells (Biological Specialty Corporation, colmar, PA) were thawed in a 37 ℃ water bath, transferred to 15mL conical tubes, and then washed once with 5mL of RPMI/10% HI FBS medium without phenol red. Cells were counted using a VIACELL XR cell viability analyzer and T cells were combined with target cells at a final effector T cell to target cell (E: T) ratio of 3:1. The cell mixtures were combined in 50mL conical tubes. The cell mixture (100. Mu.L/well) was added to a clear 96-well flat bottom plate. Next, the test antibodies were diluted in RPMI/10% HI FBS medium without phenol red to a final initial concentration of 60nM, and 3-fold serial dilutions were prepared from the initial concentration for a total of 11 dilution points. Serial dilutions of test antibodies (100 μl/well) were added to pooled cells. The plate was placed at 37℃with 5% CO ₂ A kind of electronic deviceZoom or IncuCyte +.>(Essen) for 120 hours. The target cell line stably expresses a red nuclear dye that is used to track the kinetics of target cell lysis. Percentage of inhibition of cell growthPercent) = (initial number of surviving target cells-current number of surviving target cells)/initial number of surviving cells 100%. Table 71 and FIGS. 20A-20H show cytotoxicity to C4-2B cells as the concentration of anti-PSMA increases. The isolated pan T cells were incubated with psma+c4-2B cells for 120 hours in the presence of bispecific PSMA/T cell redirecting antibodies. />

Table 71: based on Bispecific anti-PSMA/anti-T cell redirection evaluated in cytotoxicity assays of (a) Antibodies to。

Healthy PBMCs. PSMA+C4-2B human prostate tumor cells expressing red nuclear dye were generated for use in a red-basedIs described herein). Frozen vials of healthy PBMC (Hemacare, los Angeles, calif.) were thawed in a 37℃water bath, transferred to a 15mL conical tube, and washed once with 5mL phenol red free RPMI/10% HI FBS medium. Cells were counted using a VIACELL XR cell viability analyzer and PBMC were pooled with target cells at a final PBMC to target cell (E: T) ratio of 1:1. The cell mixtures were combined in 50mL conical tubes. The cell mixture (100. Mu.L/well) was added to a clear 96-well flat bottom plate. Next, the test antibodies were diluted to a final initial concentration of 30nM in RPMI/10% HI FBS medium without phenol red, and 3-fold serial dilutions were prepared from the initial concentration for a total of 11 dilution points. Serial dilutions of test antibodies (100 μl/well) were added to pooled cells. The plate was placed at 37℃with 5% CO ₂ Is->Zoom or IncuCyte +.>(Essen) for 120 hours. The target cell line stably expresses a red nuclear dye that is used to track the kinetics of target cell lysis. Percent (%) inhibition of cell growth = (initial number of target cells present-number of target cells present)/initial number of cells present 100%. FIG. 21 shows that PSMA/CD3 bispecific antibodies induced differential C4-2B cytotoxicity.

Example 10.7: evaluation of cytokine induction by bispecific anti-PSMAxCD 3 antibodies

The ability of selected bispecific PSMAxCD3 antibodies to induce cytokine release was assessed.

Supernatants collected from the in vitro cytotoxicity assays described above were analyzed using a human pro-inflammatory group I tissue culture kit (Meso Scale Discovery). The supernatant was thawed on wet ice, spun at 1,500rpm for 5 minutes at 4℃and then placed on ice. The MULT-SPOT assay plates were pre-washed according to the manufacturer's protocol. The standard curve was prepared by serial dilution of the provided calibrator in MSD diluent 1. Standards and test antibody samples (25 μl/well) were added to the pre-washed plates. Subsequent incubations and washes were performed according to the manufacturer's protocol. The assay plate was read on a SECTOR imager 6000. The ifnγ concentration of each PSMAxCD3 bispecific antibody evaluated was quantified. Figure 22 shows functional cytokine release by T cells activated by PSMAxCD3 antibodies.

Example 10.8: measurement of T cells of bispecific PSMAxCD3 antibodies to PSMA+ cells via XCELLIGENCE Mediated killing power

The ability of selected bispecific PSMAxCD3 antibodies to mediate T cell mediated killing of prostate cancer cells C4-2B was assessed. Using three pan T donors, prostate cancer cell line C4-2B expressing approximately 150,000 PSMA/cell was used at a 3:1 effector to target ratio (E: T). On day 0 of the experimentThe xcelligent plate was blank treated with 50 μl of growth medium. The plates were then inoculated with 20,000C 4-2B (0.4X10) ⁶ 50 μl of individual cells/ml) cells/well. The plate was then incubated overnight on an xcelligent machine. On day 1 of the experiment, the test was performed by adding 50. Mu.L of 1.2X10 ⁶ Three pan T donors were used to prepare the E:T ratio per mL (60,000 cells). Then 50 μl of the appropriate bispecific antibody was added to the appropriate wells of each plate. CD3xnull was used as a control. Only tumor/target wells were designated for normalization in the calculation of the percent cell lysis. The final antibody concentrations were 50nM, 10nM, 2nM, 0.4nM, 80pM and 0nM. The plate was then placed in an xcellligence machine and impedance was recorded every 15 minutes over 120 hours. Using the equation% cell lysis = [1- (NCI)/(AvgNCIR) on RTCA software ]X 100 the percent cell lysis was calculated, where NCI is the average cell index of the wells and avgcir is the average cell index of tumor reference wells alone. Table 72 summarizes cell lysis over time for each psma×cd3 bispecific molecule.

Table 72: for three pan T donors, at each dose concentration, all four bispecific antibodies were at time point 120 Summary of% cell lysis for hours。

*****

It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the present specification.

Claims

wherein the amino acid in position N106 of SEQ ID NO. 55, 54 or 48 is optionally substituted with an amino acid selected from the group consisting of A, G, S, F, E, T, R, V, I, Y, L, P, Q and K,

wherein the residue numbering starts from the N-terminus of SEQ ID NO. 55, 54 or 48.

3. The isolated protein of claim 1 or 2, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as follows:

a. SEQ ID NOs 70, 71, 72, 79, 80 and 81, respectively;

b. 70, 71, 87, 79, 80 and 81 respectively; or alternatively

c. SEQ ID NOS 70, 71, 90, 79, 80 and 81, respectively.

4. The isolated protein of claims 1-3, wherein the antigen binding domain that binds CD3 epsilon is an scFv, (scFv) 2, fv, fab, F (ab') 2, fd, dAb, or VHH.

5. The isolated protein of claim 4, wherein the antigen binding domain that binds CD3 epsilon is the Fab.

6. The isolated protein of claim 4, wherein the antigen binding domain that binds CD3 epsilon is the scFv.

7. The isolated protein of claim 6, wherein the scFv comprises VH, first linker (L1) and VL (VH-L1-VL), or VL, L1 and VH (VL-L1-VH) from N-terminus to C-terminus.

8. The isolated protein of claim 7, wherein the L1 comprises

a. About 5 to 50 amino acids;

b. about 5 to 40 amino acids;

c. about 10 to 30 amino acids; or alternatively

d. About 10 to 20 amino acids.

9. The isolated protein of claim 7, wherein the L1 comprises the amino acid sequence of SEQ ID NOs 3 to 36.

10. The isolated protein of claim 9, wherein the L1 comprises the amino acid sequence of SEQ ID No. 3.

11. The isolated protein of any one of claims 1 to 10, wherein the antigen binding domain that binds CD3 epsilon comprises the VH of SEQ ID No. 55, 54 or 48, and the VL of SEQ ID No. 59, 58 or 56.

12. The isolated protein of claim 11, wherein the antigen binding domain that binds CD3 epsilon comprises:

a VH of SEQ ID NO. 55 and VL of SEQ ID NO. 59;

VH of SEQ ID NO. 55 and VL of SEQ ID NO. 58;

VH of SEQ ID NO. 54 and VL of SEQ ID NO. 56;

the VH of SEQ ID NO. 48 and the VL of SEQ ID NO. 58;

e.VH of SEQ ID NO. 88 and VL of SEQ ID NO. 58; or alternatively

VH of SEQ ID NO:242 and VL of SEQ ID NO: 58.

13. The isolated protein of any one of claims 1-12, wherein the antigen binding domain that binds CD3 epsilon comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or 126.

14. The isolated protein of any one of claims 1 to 13, wherein the isolated protein is a multispecific protein.

15. The isolated protein of claim 14, wherein the multispecific protein is a bispecific protein.

16. The isolated protein of claim 14, wherein the multispecific protein is a trispecific protein.

17. The isolated protein of any one of claims 1-16, further comprising an immunoglobulin (Ig) constant region or a fragment of the Ig constant region.

18. The isolated protein of claim 17, wherein the fragment of the Ig constant region comprises an Fc region.

19. The isolated protein of claim 17, wherein the fragment of the Ig constant region comprises a CH2 domain.

20. The isolated protein of claim 17, wherein the fragment of the Ig constant region comprises a CH3 domain.

21. The isolated protein of claim 17, wherein the fragment of the Ig constant region comprises a CH2 domain and a CH3 domain.

22. The isolated protein of claim 17, wherein the fragment of the Ig constant region comprises at least a portion of a hinge, a CH2 domain, and a CH3 domain.

23. The isolated protein of claim 17, wherein the fragment of the Ig constant region comprises a hinge, a CH2 domain, and a CH3 domain.

24. The isolated protein of any one of claims 17-24, wherein the antigen binding domain that binds CD3 epsilon is conjugated to the N-terminus of the Ig constant region or the fragment of the Ig constant region.

25. The isolated protein of any one of claims 17-24, wherein the antigen binding domain that binds CD3 epsilon is conjugated to the C-terminus of the Ig constant region or the fragment of the Ig constant region.

26. The isolated protein of any one of claims 17-24, wherein the antigen binding domain that binds CD3 epsilon is conjugated to the Ig constant region or the fragment of the Ig constant region via a second linker (L2).

27. The isolated protein of claim 26, wherein the L2 comprises an amino acid sequence selected from the group consisting of seq id NOs 3 to 36.

28. The isolated protein of any one of claims 14 to 27, wherein the multispecific protein comprises an antigen-binding domain that binds an antigen that is not CD3 epsilon.

29. The multispecific antibody of any one of claims 14 to 28, wherein the antigen is a tumor-associated antigen.

30. The isolated protein of any one of claims 14-29, wherein the Ig constant region or the fragment of the Ig constant region is an IgG1, igG2, igG3, or IgG4 isotype.

31. The isolated protein of any one of claims 1-30, wherein the Ig constant region or the fragment of the Ig constant region comprises at least one mutation that causes reduced binding of the protein to an fcγ receptor (fcγr).

32. The isolated protein of claim 31, wherein the at least one mutation that causes reduced binding of the protein to the fcγr is selected from the group consisting of: F234A/L235A, L A/L235A, L A/L235A/D265S, V234A/G237A/P238S/H268A/V309L/A330S/P331S, F A/L235A, S P/F234A/L235A, N297A, V A/G237A, K T/E233P/L234V/L235A/G236-deletion/A327G/P331A/D365E/L358M, H268Q/V309L/A330S/P331S, S E/L328F, L F/L235E/D265A, L A/L235A/G237A/P238S/H268A/A330S/P331S, S P/F234A/L235A/G237A/P238S and S228P/F234A/L235A/G236-deletion/G237A/P238S, wherein residue numbering is performed according to the EU index.

33. The isolated protein of any one of claims 31-32, wherein the fcγr is fcγri, fcγriia, fcγriib, or fcγriii, or any combination thereof.

34. The isolated protein of any one of claims 14-33, wherein the protein comprises at least one mutation in the CH3 domain of the Ig constant region.

35. The isolated protein of claim 34, wherein the at least one mutation in the CH3 domain of the Ig constant region is selected from the group consisting of: T350V, L351Y, F A, Y407V, T366Y, T366W, T366L, T L, F405W, T394W, K392L, T394S, T394W, Y407T, Y A, T S/L368A/Y407V, L Y/F405A/Y407V, T I/K392M/T394W, F A/Y407V, T366L/K392M/T394W, T L/K392L/T394W, L57351Y/Y407A, L Y/Y407V, T A/K409F, T366V/K409F, T366A/K409F, T V/L351Y/F405A/Y407V and T350V/T366L/K392L/T394W, wherein residue numbering is according to EU index.

36. A pharmaceutical composition comprising the isolated protein of any one of claims 1 to 35, and a pharmaceutically acceptable carrier.

37. A polynucleotide encoding the isolated protein of any one of claims 1 to 35.

38. A vector comprising the polynucleotide of claim 37.

39. A host cell comprising the vector of claim 38.

40. A method of producing the isolated protein of any one of claims 1 to 35, comprising: culturing the host cell of claim 39 under conditions such that the protein is expressed, and recovering the protein produced by the host cell.

41. An anti-idiotype antibody that binds to the isolated protein of any one of claims 1 to 35.

42. The isolated protein according to any one of claims 1 to 35, comprising an amino acid sequence selected from the group consisting of seq id NOs 127 to 157.

43. The isolated protein of any one of claims 1 to 35, comprising an antibody heavy chain of SEQ ID No. 224 and an antibody light chain of SEQ ID No. 226.